[0002]The present invention relates generally to the field of molecular
biology. More specifically, the invention concerns anti-DLL4 antibodies,
and uses of same.

BACKGROUND OF THE INVENTION

[0003]Development of a vascular supply is a fundamental requirement for
many physiological and pathological processes. Actively growing tissues
such as embryos and tumors require adequate blood supply. They satisfy
this need by producing pro-angiogenic factors, which promote new blood
vessel formation via a process called angiogenesis. Vascular tube
formation is a complex but orderly biological event involving all or many
of the following steps: a) endothelial cells (ECs) proliferate from
existing ECs or differentiate from progenitor cells; b) ECs migrate and
coalesce to form cord-like structures; c) vascular cords then undergo
tubulogenesis to form vessels with a central lumen; d) existing cords or
vessels send out sprouts to form secondary vessels; e) primitive vascular
plexus undergo further remodeling and reshaping; and 1) peri-endothelial
cells are recruited to encase the endothelial tubes, providing
maintenance and modulatory functions to the vessels; such cells including
pericytes for small capillaries, smooth muscle cells for larger vessels,
and myocardial cells in the heart. Hanahan, Science 277:48-50 (1997);
Hogan & Kolodziej, Nat. Rev. Genet. 3:513-23 (2002); Lubarsky & Krasnow,
Cell 112:19-28 (2003).

[0005]In the case of tumor growth, angiogenesis appears to be crucial for
the transition from hyperplasia to neoplasia, and for providing
nourishment for the growth and metastasis of the tumor. Folkman et al.,
Nature 339:58 (1989). The neovascularization allows the tumor cells to
acquire a growth advantage and proliferative autonomy compared to the
normal cells. A tumor usually begins as a single aberrant cell which can
proliferate only to a size of a few cubic millimeters due to the distance
from available capillary beds, and it can stay `dormant` without further
growth and dissemination for a long period of time. Some tumor cells then
switch to the angiogenic phenotype to activate endothelial cells, which
proliferate and mature into new capillary blood vessels. These newly
formed blood vessels not only allow for continued growth of the primary
tumor, but also for the dissemination and recolonization of metastatic
tumor cells. Accordingly, a correlation has been observed between density
of microvessels in tumor sections and patient survival in breast cancer
as well as in several other tumors. Weidner et al., N Engl. J. Med.
324:1-6 (1991); Horak et al., Lancet 340:1120-24 (1992); Macchiarini et
al., Lancet 340:145-46 (1992). The precise mechanisms that control the
angiogenic switch is not well understood, but it is believed that
neovascularization of tumor mass results from the net balance of a
multitude of angiogenesis stimulators and inhibitors (Folkman, Nat. Med.
1(1):27-31 (1995)).

[0006]The process of vascular development is tightly regulated. To date, a
significant number of molecules, mostly secreted factors produced by
surrounding cells, have been shown to regulate EC differentiation,
proliferation, migration and coalescence into cord-like structures. For
example, vascular endothelial growth factor (VEGF) has been identified as
the key factor involved in stimulating angiogenesis and in inducing
vascular permeability. Ferrara et al., Endocr. Rev. 18:4-25 (1997). The
finding that the loss of even a single VEGF allele results in embryonic
lethality points to an irreplaceable role played by this factor in the
development and differentiation of the vascular system. Furthermore, VEGF
has been shown to be a key mediator of neovascularization associated with
tumors and intraocular disorders. Ferrara et al., Endocr. Rev. supra. The
VEGF mRNA is overexpressed by the majority of human tumors examined.
Berkman et al., J. Clin. Invest. 91:153-59 (1993); Brown et al., Human
Pathol. 26:86-91 (1995); Brown et al., Cancer Res. 53:4727-35 (1993);
Mattern et al., Brit. J. Cancer 73:931-34 (1996); Dvorak et al., Am. J.
Pathol. 146:1029-39 (1995).

[0008]Anti-VEGF neutralizing antibodies suppress the growth of a variety
of human tumor cell lines in nude mice (Kim et al., Nature 362:841-44
(1993); Warren et al., J. Clin. Invest. 95:1789-97 (1995); Borgstrom et
al., Cancer Res. 56:4032-39 (1996); Melnyk et al., Cancer Res. 56:921-24
(1996)) and also inhibit intraocular angiogenesis in models of ischemic
retinal disorders (Adamis et al., Arch. Opthalmol. 114:66-71 (1996)).
Therefore, anti-VEGF monoclonal antibodies or other inhibitors of VEGF
action are promising candidates for the treatment of tumors and various
intraocular neovascular disorders. Such antibodies are described, for
example, in EP 817,648, published Jan. 14, 1998; and in WO 98/45331 and
WO 98/45332, both published Oct. 15, 1998. One anti-VEGF antibody,
bevacizumab, has been approved by the FDA for use in combination with a
chemotherapy regimen to treat specific cancers and an anti-VEGF antibody
fragment, ranibizumab, has been approved by the FDA to treat age-related
(wet) macular degeneration. Both drugs are being investigated in ongoing
clinical trials.

[0009]It is clear that there continues to be a need for agents that have
clinical attributes that are optimal for development as therapeutic
agents. The invention described herein meets this need and provides other
benefits.

[0010]All references cited herein, including patent applications and
publications, are incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

[0011]The invention is in part based on the identification of a variety of
DLL4 binding agents (such as immunoconjugates, antibodies, and fragments
thereof). DLL4 presents as an important and advantageous therapeutic
target, and the invention provides compositions and methods based on
binding DLL4. DLL4 binding agents of the invention, as described herein,
provide important therapeutic and diagnostic agents for use in targeting
pathological conditions associated with expression and/or activity of the
DLL4-Notch receptor pathways. Accordingly, the invention provides
methods, compositions, kits and articles of manufacture related to DLL4
binding.

[0013]In one aspect, the invention provides an isolated anti-DLL4
antibody, wherein a full length IgG form of the antibody specifically
binds human DLL4 with a binding affinity of about 1 nM or better, or
about 500 μM or better. As is well-established in the art, binding
affinity of a ligand to its receptor can be determined using any of a
variety of assays, and expressed in terms of a variety of quantitative
values. Accordingly, in one embodiment, the binding affinity is expressed
as Kd values and reflects intrinsic binding affinity (e.g., with
minimized avidity effects). Generally and preferably, binding affinity is
measured in vitro, whether in a cell-free or cell-associated setting. Any
of a number of assays known in the art, including those described herein,
can be used to obtain binding affinity measurements, including, for
example, Biacore®, radioimmunoassay (RIA) and ELISA. In some
embodiments, the isolated anti-DLL4 antibody binds to both human and
mouse DLL4 with similar affinity, i.e. the binding affinity for human
DLL4 is no more than 100-fold more or less than the binding affinity for
mouse DLL4. In some embodiments, the binding affinity for human DLL4 is
no more than 10-fold more or less than the binding affinity for mouse
DLL4. In some embodiments, the antibody specifically binds mouse DLL4
with a binding affinity of about 1 nM or better, or about 500 pM or
better.

[0014]In one aspect, the invention provides an isolated anti-DLL4
antibody, wherein a full length IgG form of the antibody specifically
binds human DLL4 with a kon of about 2×105 or better, or
about 1×105 or better. As is well-established in the art, the
kon of binding of a ligand to its receptor can be determined using
any of a variety of assays and expressed in terms of a variety of
quantitative values.

[0015]In one aspect, the invention provides an isolated antibody that
binds a ligand binding region of DLL4. In some embodiments, the isolated
antibody binds a polypeptide comprising, consisting of or consisting
essentially of the DLL4 extracellular domain. In some embodiments, the
isolated antibody binds a polypeptide comprising, consisting of or
consisting essentially of amino acids 252-282, 1-252, 1-286, 1-324,
and/or 219-286 of human DLL4.

[0016]In one aspect, the invention provides an isolated anti-DLL4 antibody
that competes with Notch receptor for binding of DLL4.

[0048]In one embodiment, a HVR-H2 variant comprises 1-4 (1, 2, 3, or 4)
substitutions in any combination of the following positions: 50 (V, L or
Y), 52 (N or S), 52a (P or S), or 53 (N, Q, T, or I).

[0049]Letter(s) in parenthesis following each position indicates an
illustrative substitution (i.e., replacement) amino acid; as would be
evident to one skilled in the art, suitability of other amino acids as
substitution amino acids in the context described herein can be routinely
assessed using techniques known in the art and/or described herein.

[0050]In one aspect, the invention provides an antibody comprising a
HVR-H1 region comprising the sequence of SEQ ID NO:1 or 2. In one aspect,
the invention provides an antibody comprising a HVR-H2 region comprising
the sequence of SEQ ID NO:3, 4, 5, 6, 7, or 8. In one aspect, the
invention provides an antibody comprising a HVR-H3 region comprising the
sequence of SEQ ID NO: 9. In one embodiment, the invention provides an
antibody comprising a HVR-L1 region comprising the sequence of SEQ ID
NO:10. In one embodiment, the invention provides an antibody comprising a
HVR-L2 region comprising the sequence of SEQ ID NO: 11. In one
embodiment, the invention provides an antibody comprising a HVR-L3 region
comprising the sequence of SEQ ID NO: 12, 13, 14, 15, 16, 17, or 18.

[0051]In one aspect, the invention provides an antibody comprising at
least one, at least two, or all three of the following:

[0067]The amino acid sequences of SEQ ID NOs:1-18 are numbered with
respect to individual HVR (i.e., H1, H2 or H3) as indicated in FIGS. 1a
and 1b, the numbering being consistent with the Kabat numbering system as
described below.

[0071]In one embodiment, the huMAb4D5-8 light chain variable domain
sequence is modified at one or more of positions 30, 66 and 91 (Asn, Arg
and H is as indicated in bold/italics above, respectively). In one
embodiment, the modified huMAb4D5-8 sequence comprises Ser in position
30, Gly in position 66 and/or Ser in position 91. Accordingly, in one
embodiment, an antibody of the invention comprises a light chain variable
domain comprising the sequence depicted in SEQ ID NO:53 below:

[0079]In one embodiment, an antibody of the invention is affinity matured
to obtain the target binding affinity desired. In one example, an
affinity matured antibody of the invention comprises substitution at one
or more of amino acid position H28, H30, H31, H32, H33, L91, L92, L93,
L94, L95 and/or L96. In one example an affinity matured antibody of the
invention comprises one or more of the following substitutions: (a) in
the heavy chain, V50L, V50Y, N52S, P52aS, N53Q, N53T, N53I, S56A, S56F,
T57S, D58E, D58I, D58A, D58Y, or (b), in the light chain, S91W, Y92F,
T93N, T93S, T94G, P95Q, P95A, P95T, P96S, P96A, P96V.

[0080]In one embodiment, an antibody of the invention comprises a heavy
chain variable domain comprising the sequence of SEQ ID NO:54. In one
embodiment, an antibody of the invention comprises a light chain variable
domain comprising the sequence of SEQ ID NO:55. In one embodiment, an
antibody of the invention comprises a heavy chain variable domain
comprising the sequence of SEQ ID NO:54 and a light chain variable domain
comprising the sequence of SEQ ID NO:55.

[0081]In one embodiment, an antibody of the invention comprises a heavy
chain variable domain comprising the sequence of SEQ ID NO:56. In one
embodiment, an antibody of the invention comprises a light chain variable
domain comprising the sequence of SEQ ID NO:57. In one embodiment, an
antibody of the invention comprises a heavy chain variable domain
comprising the sequence of SEQ ID NO:56 and a light chain variable domain
comprising the sequence of SEQ ID NO:57.

[0082]In one embodiment, an antibody of the invention comprises a heavy
chain variable domain comprising the sequence of SEQ ID NO:58. In one
embodiment, an antibody of the invention comprises a light chain variable
domain comprising the sequence of SEQ ID NO:59. In one embodiment, an
antibody of the invention comprises a heavy chain variable domain
comprising the sequence of SEQ ID NO:58 and a light chain variable domain
comprising the sequence of SEQ ID NO:59.

[0083]In one aspect, the invention provides an antibody that competes with
any of the above-mentioned antibodies for binding to DLL4. In one aspect,
the invention provides an antibody that binds to the same epitope on DLL4
as any of the above-mentioned antibodies.

[0084]As is known in the art, and as described in greater detail
hereinbelow, the amino acid position/boundary delineating a hypervariable
region of an antibody can vary, depending on the context and the various
definitions known in the art (as described below). Some positions within
a variable domain may be viewed as hybrid hypervariable positions in that
these positions can be deemed to be within a hypervariable region under
one set of criteria while being deemed to be outside a hypervariable
region under a different set of criteria. One or more of these positions
can also be found in extended hypervariable regions (as further defined
below).

[0085]In some embodiments, the antibody is a monoclonal antibody. In some
embodiments, the antibody is a polyclonal antibody. In some embodiments,
the antibody is selected from the group consisting of a chimeric
antibody, an affinity matured antibody, a humanized antibody, and a human
antibody. In some embodiments, the antibody is an antibody fragment. In
some embodiments, the antibody is a Fab, Fab', Fab'-SH, F(ab')2, or
scFv.

[0086]In one embodiment, the antibody is a chimeric antibody, for example,
an antibody comprising antigen binding sequences from a non-human donor
grafted to a heterologous non-human, human or humanized sequence (e.g.,
framework and/or constant domain sequences). In one embodiment, the
non-human donor is a mouse. In one embodiment, an antigen binding
sequence is synthetic, e.g. obtained by mutagenesis (e.g., phage display
screening, etc.). In one embodiment, a chimeric antibody of the invention
has murine V regions and human C region. In one embodiment, the murine
light chain V region is fused to a human kappa light chain. In one
embodiment, the murine heavy chain V region is fused to a human IgG1 C
region.

[0087]Humanized antibodies of the invention include those that have amino
acid substitutions in the FR and affinity maturation variants with
changes in the grafted CDRs. The substituted amino acids in the CDR or FR
are not limited to those present in the donor or recipient antibody. In
other embodiments, the antibodies of the invention further comprise
changes in amino acid residues in the Fc region that lead to improved
effector function including enhanced CDC and/or ADCC function and B-cell
killing. Other antibodies of the invention include those having specific
changes that improve stability. In other embodiments, the antibodies of
the invention comprise changes in amino acid residues in the Fc region
that lead to decreased effector function, e.g. decreased CDC and/or ADCC
function and/or decreased B-cell killing. In some embodiments, the
antibodies of the invention are characterized by decreased binding (such
as absence of binding) to human complement factor C1q and/or human Fc
receptor on natural killer (NK) cells. In some embodiments, the
antibodies of the invention are characterized by decreased binding (such
as the absence of binding) to human FcγRI, FcγRIIA, and/or
FcγRIIIA. In some embodiments, the antibodies of the invention is
of the IgG class (eg, IgG1 or IgG4) and comprises at least one mutation
in E233, L234, G236, D265, D270, N297, E318, K320, K322, A327,
A330, P331 and/or P329 (numbering according to the EU index). In some
embodiments, the antibodies comprise the mutation L234A/L235A or
D265A/N297A.

[0089]The antibodies of the invention bind (such as specifically bind)
DLL4, and in some embodiments, may modulate one or more aspects of
DLL4-associated effects, including but not limited to any one or more of
reduction or blocking of Notch receptor activation, reduction or blocking
of Notch receptor downstream molecular signaling, disruption or blocking
of Notch receptor binding to DLL4, and/or promotion of endothelial cell
proliferation, and/or inhibition of endothelial cell differentiation,
and/or inhibition of arterial differentiation, and/or inhibition of tumor
vascular perfusion, and/or treatment and/or prevention of a tumor, cell
proliferative disorder or a cancer; and/or treatment or prevention of a
disorder associated with DLL4 expression and/or activity and/or treatment
or prevention of a disorder associated with Notch receptor expression
and/or activity. In some embodiments, the antibody of the invention
specifically binds to DLL4. In some embodiments, the antibody
specifically binds to the DLL4 extracellular domain (ECD). In some
embodiments, the antibody specifically binds to a polypeptide consisting
of or consisting essentially of the DLL4 extracellular domain. In some
embodiments, the antibody specifically binds DLL4 with a KD of about 1 nM
or better, or about 500 μM or better. In some embodiments, the
antibody specifically binds human DLL4 with a kon of about
2×105 or better, or about 1×105 or better. In some
embodiments, the antibody of the invention reduces, inhibits, and/or
blocks DLL4 activity in vivo and/or in vitro. In some embodiments, the
antibody competes for binding with DLL4-ligand (reduces and/or blocks
Notch receptor binding to DLL4).

[0090]In one aspect, the invention provides compositions comprising one or
more antibodies of the invention and a carrier. In one embodiment, the
carrier is pharmaceutically acceptable.

[0091]In one aspect, the invention provides nucleic acids encoding an
anti-DLL4 antibody of the invention.

[0092]In one aspect, the invention provides vectors comprising a nucleic
acid of the invention.

[0093]In one aspect, the invention provides compositions comprising one or
more nucleic acid of the invention and a carrier. In one embodiment, the
carrier is pharmaceutically acceptable.

[0094]In one aspect, the invention provides host cells comprising a
nucleic acid or a vector of the invention. A vector can be of any type,
for example a recombinant vector such as an expression vector. Any of a
variety of host cells can be used. In one embodiment, a host cell is a
prokaryotic cell, for example, E. coli. In one embodiment, a host cell is
a eukaryotic cell, for example a mammalian cell such as Chinese Hamster
Ovary (CHO) cell.

[0095]In one aspect, the invention provides methods of making an antibody
of the invention. For example, the invention provides methods of making
an anti-DLL4 antibody (which, as defined herein includes full length and
fragments thereof) or immunoconjugate, said method comprising expressing
in a suitable host cell a recombinant vector of the invention encoding
said antibody (or fragment thereof), and recovering said antibody.

[0096]In one aspect, the invention provides an article of manufacture
comprising a container; and a composition contained within the container,
wherein the composition comprises one or more anti-DLL4 antibodies of the
invention. In one embodiment, the composition further comprises an
anti-angiogenesis agent. In one embodiment the anti-angiogenesis agent is
an anti-VEGF antibody, e.g., bevacizumab. In one embodiment, the
composition comprises a nucleic acid of the invention. In one embodiment,
a composition comprising an antibody further comprises a carrier, which
in some embodiments is pharmaceutically acceptable. In one embodiment, a
second composition is contained within the container, wherein the second
composition comprises an anti-angiogenesis agent. In one embodiment, the
anti-angiogenesis agent is an anti-VEGF antibody, e.g. bevacizumab. In
one embodiment, an article of manufacture of the invention further
comprises instructions for administering the composition(s) (for e.g.,
the antibody) to a subject (such as instructions for any of the methods
described herein).

[0097]In one aspect, the invention provides a kit comprising a first
container comprising a composition comprising one or more anti-DLL4
antibodies of the invention; and a second container comprising a buffer.
In one embodiment, the buffer is pharmaceutically acceptable. In one
embodiment, a composition comprising an antibody further comprises a
carrier, which in some embodiments is pharmaceutically acceptable. In one
embodiment, a kit further comprises instructions for administering the
composition (for e.g., the antibody) to a subject.

[0098]In one aspect, the invention provides use of an anti-DLL4 antibody
of the invention in the preparation of a medicament for the therapeutic
and/or prophylactic treatment of a disorder, such as a cancer, a tumor,
and/or a cell proliferative disorder. In some embodiments, the disorder
is a pathological condition associated with angiogenesis.

[0099]In one aspect, the invention provides use of a nucleic acid of the
invention in the preparation of a medicament for the therapeutic and/or
prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a
cell proliferative disorder. In some embodiments, the disorder is a
pathological condition associated with angiogenesis.

[0100]In one aspect, the invention provides use of an expression vector of
the invention in the preparation of a medicament for the therapeutic
and/or prophylactic treatment of a disorder, such as a cancer, a tumor,
and/or a cell proliferative disorder. In some embodiments, the disorder
is a pathological condition associated with angiogenesis.

[0101]In one aspect, the invention provides use of a host cell of the
invention in the preparation of a medicament for the therapeutic and/or
prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a
cell proliferative disorder. In some embodiments, the disorder is a
pathological condition associated with angiogenesis.

[0102]In one aspect, the invention provides use of an article of
manufacture of the invention in the preparation of a medicament for the
therapeutic and/or prophylactic treatment of a disorder, such as a
cancer, a tumor, and/or a cell proliferative disorder. In some
embodiments, the disorder is a pathological condition associated with
angiogenesis.

[0103]In one aspect, the invention provides use of a kit of the invention
in the preparation of a medicament for the therapeutic and/or
prophylactic treatment of a disorder, such as a cancer, a tumor, and/or a
cell proliferative disorder. In some embodiments, the disorder is a
pathological condition associated with angiogenesis.

[0105]In one aspect, the invention provides methods for treating or
preventing a tumor, a cancer, and/or a cell proliferative disorder
associated with increased expression and/or activity of DLL4, the methods
comprising administering an effective amount of an anti-DLL4 antibody to
a subject in need of such treatment.

[0106]In one aspect, the invention provides methods for reducing,
inhibiting, blocking, or preventing growth of a tumor or cancer, the
methods comprising administering an effective amount of an anti-DLL4
antibody to a subject in need of such treatment.

[0107]In one aspect, the invention provides methods for treating a tumor,
a cancer, and/or a cell proliferative disorder comprising administering
an effective amount of an anti-DLL4 antibody to a subject in need of such
treatment.

[0108]In one aspect, the invention provides methods for inhibiting
angiogenesis comprising administering an effective amount of an anti-DLL4
antibody to a subject in need of such treatment.

[0109]In one aspect, the invention provides methods for treating a
pathological condition associated with angiogenesis comprising
administering an effective amount of an anti-DLL4 antibody to a subject
in need of such treatment. In some embodiments, the pathological
condition associated with angiogenesis is a tumor, a cancer, and/or a
cell proliferative disorder. In some embodiments, the pathological
condition associated with angiogenesis is an intraocular neovascular
disease.

[0110]Methods of the invention can be used to affect any suitable
pathological state. Exemplary disorders are described herein, and include
cancers selected from the group consisting of small cell lung cancer,
neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal
cancer (CRC), and hepatocellular carcinoma, including metastatic forms of
those cancers.

[0111]Methods of the invention can further comprise additional treatment
steps. For example, in one embodiment, a method further comprises a step
wherein a targeted cell and/or tissue (for e.g., a cancer cell) is
exposed to radiation treatment or a chemotherapeutic agent or an
anti-angiogenic agent.

[0112]In another aspect, the invention provides methods for detection of
DLL4, the methods comprising detecting DLL4-anti-DLL4 antibody complex in
the sample. The term "detection" as used herein includes qualitative
and/or quantitative detection (measuring levels) with or without
reference to a control.

[0113]In another aspect, the invention provides methods for diagnosing a
disorder associated with DLL4 expression and/or activity, the methods
comprising detecting DLL4-anti-DLL4 antibody complex in a biological
sample from a patient having or suspected of having the disorder. In some
embodiments, the DLL4 expression is increased expression or abnormal
expression. In some embodiments, the disorder is a tumor, cancer, and/or
a cell proliferative disorder.

[0114]In another aspect, the invention provides any of the anti-DLL4
antibodies described herein, wherein the anti-DLL4 antibody comprises a
detectable label.

[0115]In another aspect, the invention provides a complex of any of the
anti-DLL4 antibodies described herein and DLL4. In some embodiments, the
complex is in vivo or in vitro. In some embodiments, the complex
comprises a cancer cell. In some embodiments, the anti-DLL4 antibody is
detectably labeled.

[0130]The invention herein provides anti-DLL4 antibodies, that are useful
for, e.g., treatment or prevention of disease states associated with
expression and/or activity of DLL4, such as increased expression and/or
activity or undesired expression and/or activity. In some embodiments,
the antibodies of the invention are used to treat a tumor, a cancer,
and/or a cell proliferative disorder. In some embodiments, the antibodies
of the invention are used to treat a pathological condition associated
with angiogenesis.

[0131]In another aspect, the anti-DLL4 antibodies of the invention find
utility as reagents for detection and/or isolation of DLL4, such as
detection of DLL4 in various tissues and cell type.

[0134]An "isolated" antibody is one which has been identified and
separated and/or recovered from a component of its natural environment.
Contaminant components of its natural environment are materials which
would interfere with diagnostic or therapeutic uses for the antibody, and
may include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. In preferred embodiments, the antibody will be
purified (1) to greater than 95% by weight of antibody as determined by
the Lowry method, and most preferably more than 99% by weight, (2) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator, or (3)
to homogeneity by SDS-PAGE under reducing or nonreducing conditions using
Coomassie® blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at least one
component of the antibody's natural environment will not be present.
Similarly, isolated antibody includes the antibody in medium around
recombinant cells. Ordinarily, however, isolated antibody will be
prepared by at least one purification step.

[0135]An "isolated" nucleic acid molecule is a nucleic acid molecule that
is identified and separated from at least one contaminant nucleic acid
molecule with which it is ordinarily associated in the natural source of
the antibody nucleic acid. An isolated nucleic acid molecule is other
than in the form or setting in which it is found in nature. Isolated
nucleic acid molecules therefore are distinguished from the nucleic acid
molecule as it exists in natural cells. However, an isolated nucleic acid
molecule includes a nucleic acid molecule contained in cells that
ordinarily express the antibody where, for example, the nucleic acid
molecule is in a chromosomal location different from that of natural
cells.

[0136]The term "variable domain residue numbering as in Kabat" or "amino
acid position numbering as in Kabat", and variations thereof, refers to
the numbering system used for heavy chain variable domains or light chain
variable domains of the compilation of antibodies in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public Health
Service, National Institutes of Health, Bethesda, Md. (1991). Using this
numbering system, the actual linear amino acid sequence may contain fewer
or additional amino acids corresponding to a shortening of, or insertion
into, a FR or CDR of the variable domain. For example, a heavy chain
variable domain may include a single amino acid insert (residue 52a
according to Kabat) after residue 52 of H2 and inserted residues (e.g.
residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR
residue 82. The Kabat numbering of residues may be determined for a given
antibody by alignment at regions of homology of the sequence of the
antibody with a "standard" Kabat numbered sequence.

[0137]The phrase "substantially similar," or "substantially the same", as
used herein, denotes a sufficiently high degree of similarity between two
numeric values (generally one associated with an antibody of the
invention and the other associated with a reference/comparator antibody)
such that one of skill in the art would consider the difference between
the two values to be of little or no biological and/or statistical
significance within the context of the biological characteristic measured
by said values (e.g., Kd values). The difference between said two values
is preferably less than about 50%, preferably less than about 40%,
preferably less than about 30%, preferably less than about 20%,
preferably less than about 10% as a function of the value for the
reference/comparator antibody.

[0138]"Binding affinity" generally refers to the strength of the sum total
of noncovalent interactions between a single binding site of a molecule
(e.g., an antibody) and its binding partner (e.g., an antigen). Unless
indicated otherwise, as used herein, "binding affinity" refers to
intrinsic binding affinity which reflects a 1:1 interaction between
members of a binding pair (e.g., antibody and antigen). The affinity of a
molecule X for its partner Y can generally be represented by the
dissociation constant (Kd). Affinity can be measured by common methods
known in the art, including those described herein. Low-affinity
antibodies generally bind antigen slowly and tend to dissociate readily,
whereas high-affinity antibodies generally bind antigen faster and tend
to remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for purposes of
the present invention. Specific illustrative embodiments are described in
the following.

[0139]In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of an antibody of interest and its antigen
as described by the following assay that measures solution binding
affinity of Fabs for antigen by equilibrating Fab with a minimal
concentration of (125B-labeled antigen in the presence of a titration
series of unlabeled antigen, then capturing bound antigen with an
anti-Fab antibody-coated plate (Chen, et al., (1999) J. Mol. Biol
293:865-881). To establish conditions for the assay, microtiter plates
(Dynex) are coated overnight with 5 ug/ml of a capturing anti-Fab
antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and
subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to
five hours at room temperature (approximately 23° C.). In a
non-adsorbant plate (Nunc #269620), 100 μM or 26 μM
[125I]-antigen are mixed with serial dilutions of a Fab of interest
(e.g., consistent with assessment of an anti-VEGF antibody, Fab-12, in
Presta et al., (1997) Cancer Res. 57:4593-4599). The Fab of interest is
then incubated overnight; however, the incubation may continue for a
longer period (e.g., 65 hours) to insure that equilibrium is reached.
Thereafter, the mixtures are transferred to the capture plate for
incubation at room temperature (e.g., for one hour). The solution is then
removed and the plate washed eight times with 0.1% Tween®20 in PBS.
When the plates have dried, 150 μl/well of scintillant
(MicroScint®-20; Packard) is added, and the plates are counted on a
TopCount gamma counter (Packard) for ten minutes. Concentrations of each
Fab that give less than or equal to 20% of maximal binding are chosen for
use in competitive binding assays. According to another embodiment the Kd
or Kd value is measured by using surface plasmon resonance assays using a
BIAcore®-2000 or a BIAcore®-3000 (BIAcore, Inc., Piscataway, N.J.)
at 25° C. with immobilized antigen CM5 chips at ˜10 response
units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5,
BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and
N-hydroxysuccinimide (NHS) according to the supplier's instructions.
Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 μg/ml
(˜0.2 μM) before injection at a flow rate of 5 μl/minute to
achieve approximately 10 response units (RU) of coupled protein.
Following the injection of antigen, 1M ethanolamine is injected to block
unreacted groups. For kinetics measurements, two-fold serial dilutions of
Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% Tween®20
(PBST) at 25° C. at a flow rate of approximately 25 μl/min.
Association rates (kon) and dissociation rates (koff) are
calculated using a simple one-to-one Langmuir binding model (BIAcore
Evaluation Software version 3.2) by simultaneous fitting the association
and dissociation sensorgram. The equilibrium dissociation constant (Kd)
is calculated as the ratio koff/kon. See, e.g., Chen, Y., et
al., (1999) J. Mol. Biol 293:865-881. If the on-rate exceeds 106M-1
S-1 by the surface plasmon resonance assay above, then the on-rate
can be determined by using a fluorescent quenching technique that
measures the increase or decrease in fluorescence emission intensity
(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of
a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence
of increasing concentrations of antigen as measured in a spectrometer,
such as a stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a stir
red cuvette.

[0140]An "on-rate" or "rate of association" or "association rate" or
"kon" according to this invention can also be determined with the
same surface plasmon resonance technique described above using a
BIAcore®-2000 or a BIAcore®-3000 (BIAcore, Inc., Piscataway, N.J.)
at 25 C with immobilized antigen CMS chips at ˜10 response units
(RU). Briefly, carboxymethylated dextran biosensor chips (CMS, BIAcore
Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH
4.8, into 5 μg/ml (˜0.2 μM) before injection at a flow rate
of 5 ul/minute to achieve approximately 10 response units (RU) of coupled
protein. Following the injection of antigen, 1M ethanolamine is injected
to block unreacted groups. For kinetics measurements, two-fold serial
dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%
Tween®20 (PBST) at 25° C. at a flow rate of approximately 25
μl/min. Association rates (kon) and dissociation rates
(koff) are calculated using a simple one-to-one Langmuir binding
model (BIAcore Evaluation Software version 3.2) by simultaneous fitting
the association and dissociation sensorgram. The equilibrium dissociation
constant (Kd) was calculated as the ratio koff/kon. See, e.g.,
Chen, Y., et al., (1999) J. Mol Biol 293:865-881. However, if the on-rate
exceeds 106 M-1 S-1 by the surface plasmon resonance assay
above, then the on-rate is preferably determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16
nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations of
antigen as measured in a a spectrometer, such as a stop-flow equipped
spectrophometer (Aviv Instruments) or a 8000-series SLM-Aminco
spectrophotometer (ThermoSpectronic) with a stirred cuvette.

[0141]The term "vector," as used herein, is intended to refer to a nucleic
acid molecule capable of transporting another nucleic acid to which it
has been linked One type of vector is a "plasmid", which refers to a
circular double stranded DNA loop into which additional DNA segments may
be ligated. Another type of vector is a phage vector. Another type of
vector is a viral vector, wherein additional DNA segments may be ligated
into the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)
can be integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of
genes to which they are operatively linked. Such vectors are referred to
herein as "recombinant expression vectors" (or simply, "recombinant
vectors"). In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" may, at times, be used
interchangeably as the plasmid is the most commonly used form of vector.

[0142]"Polynucleotide," or "nucleic acid," as used interchangeably herein,
refer to polymers of nucleotides of any length, and include DNA and RNA.
The nucleotides can be deoxyribonucleotides, ribonucleotides, modified
nucleotides or bases, and/or their analogs, or any substrate that can be
incorporated into a polymer by DNA or RNA polymerase, or by a synthetic
reaction. A polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification to the
nucleotide structure may be imparted before or after assembly of the
polymer. The sequence of nucleotides may be interrupted by non-nucleotide
components. A polynucleotide may be further modified after synthesis,
such as by conjugation with a label. Other types of modifications
include, for example, "caps", substitution of one or more of the
naturally occurring nucleotides with an analog, internucleotide
modifications such as, for example, those with uncharged linkages (e.g.,
methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates, phosphorodithioates,
etc.), those containing pendant moieties, such as, for example, proteins
(e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine,
etc.), those with intercalators (e.g., acridine, psoralen, etc.), those
containing chelators (e.g., metals, radioactive metals, boron, oxidative
metals, etc.), those containing alkylators, those with modified linkages
(e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms
of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups, or
activated to prepare additional linkages to additional nucleotides, or
may be conjugated to solid or semi-solid supports. The 5' and 3' terminal
OH can be phosphorylated or substituted with amines or organic capping
group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be
derivatized to standard protecting groups. Polynucleotides can also
contain analogous forms of ribose or deoxyribose sugars that are
generally known in the art, including, for example, 2'-O-methyl-,
2'-O-allyl, 2'-fluoro- or 2'-azido-ribose, carbocyclic sugar analogs,
alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses or
lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic
analogs and a basic nucleoside analogs such as methyl riboside. One or
more phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not limited to,
embodiments wherein phosphate is replaced by P(O)S("thioate"), P(S)S
("dithioate"), "(O)NR2 ("amidate"), P(O)R, P(O)OR', CO or CH2
("formacetal"), in which each R or R' is independently H or substituted
or unsubstituted alkyl (1-20 C) optionally containing an ether (--O--)
linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all
linkages in a polynucleotide need be identical. The preceding description
applies to all polynucleotides referred to herein, including RNA and DNA.

[0143]"Oligonucleotide," as used herein, generally refers to short,
generally single stranded, generally synthetic polynucleotides that are
generally, but not necessarily, less than about 200 nucleotides in
length. The terms "oligonucleotide" and "polynucleotide" are not mutually
exclusive. The description above for polynucleotides is equally and fully
applicable to oligonucleotides.

[0144]"Percent (%) amino acid sequence identity" with respect to a peptide
or polypeptide sequence is defined as the percentage of amino acid
residues in a candidate sequence that are identical with the amino acid
residues in the specific peptide or polypeptide sequence, after aligning
the sequences and introducing gaps, if necessary, to achieve the maximum
percent sequence identity, and not considering any conservative
substitutions as part of the sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance, using
publicly available computer software such as BLAST, BLAST-2, ALIGN or
MegAlign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any algorithms
needed to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison computer
program ALIGN-2. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. and the source code has been filed with user
documentation in the U.S. Copyright Office, Washington D.C., 20559, where
it is registered under U.S. Copyright Registration No. TXU510087. The
ALIGN-2 program is publicly available through Genentech, Inc., South San
Francisco, Calif. The ALIGN-2 program should be compiled for use on a
UNIX operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not vary.

[0145]In situations where ALIGN-2 is employed for amino acid sequence
comparisons, the % amino acid sequence identity of a given amino acid
sequence A to, with, or against a given amino acid sequence B (which can
alternatively be phrased as a given amino acid sequence A that has or
comprises a certain % amino acid sequence identity to, with, or against a
given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matches
by the sequence alignment program ALIGN-2 in that program's alignment of
A and B, and where Y is the total number of amino acid residues in B. It
will be appreciated that where the length of amino acid sequence A is not
equal to the length of amino acid sequence B, the % amino acid sequence
identity of A to B will not equal the % amino acid sequence identity of B
to A.

[0146]Unless specifically stated otherwise, all % amino acid sequence
identity values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.

[0148]The term "Notch receptor" (interchangeably termed "Notch"), as used
herein, refers, unless specifically or contextually indicated otherwise,
to any native or variant (whether native or synthetic) Notch receptor
polypeptide. Humans have four Notch receptors (Notch1, Notch 2, Notch3,
and Notch4). As used herein, the term Notch receptor includes any one of
or all four human Notch receptors. The term "native sequence"
specifically encompasses naturally occurring truncated or secreted forms
(e.g., an extracellular domain sequence), naturally occurring variant
forms (e.g., alternatively spliced forms) and naturally-occurring allelic
variants. The term "wild type Notch receptor" generally refers to a
polypeptide comprising the amino acid sequence of a naturally occurring
Notch receptor protein. The term "wild type Notch receptor sequence"
generally refers to an amino acid sequence found in a naturally occurring
Notch receptor.

[0149]The terms "antibody" and "immunoglobulin" are used interchangeably
in the broadest sense and include monoclonal antibodies (e.g., full
length or intact monoclonal antibodies), polyclonal antibodies,
multivalent antibodies, multispecific antibodies (e.g., bispecific
antibodies so long as they exhibit the desired biological activity) and
may also include certain antibody fragments (as described in greater
detail herein). An antibody can be human, humanized and/or affinity
matured.

[0150]The term "variable" refers to the fact that certain portions of the
variable domains differ extensively in sequence among antibodies and are
used in the binding and specificity of each particular antibody for its
particular antigen. However, the variability is not evenly distributed
throughout the variable domains of antibodies. It is concentrated in
three segments called complementarity-determining regions (CDRs) or
hypervariable regions (HVRs) both in the light-chain and the heavy-chain
variable domains. The more highly conserved portions of variable domains
are called the framework (FR). The variable domains of native heavy and
light chains each comprise four FR regions, largely adopting a
β-sheet configuration, connected by three CDRs, which form loops
connecting, and in some cases forming part of, the β-sheet
structure. The CDRs in each chain are held together in close proximity by
the FR regions and, with the CDRs from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et al.,
Sequences of Proteins of Immunological Interest, Fifth Edition, National
Institute of Health, Bethesda, Md. (1991)). The constant domains are not
involved directly in binding an antibody to an antigen, but exhibit
various effector functions, such as participation of the antibody in
antibody-dependent cellular toxicity.

[0151]Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, each with a single
antigen-binding site, and a residual "Fc" fragment, whose name reflects
its ability to crystallize readily. Pepsin treatment yields an
F(ab')2 fragment that has two antigen-combining sites and is still
capable of cross-linking antigen.

[0152]"Fv" is the minimum antibody fragment which contains a complete
antigen-recognition and -binding site. In a two-chain Fv species, this
region consists of a dimer of one heavy- and one light-chain variable
domain in tight, non-covalent association. In a single-chain Fv species,
one heavy- and one light-chain variable domain can be covalently linked
by a flexible peptide linker such that the light and heavy chains can
associate in a "dimeric" structure analogous to that in a two-chain Fv
species. It is in this configuration that the three CDRs of each variable
domain interact to define an antigen-binding site on the surface of the
VH-VL dimer. Collectively, the six CDRs confer antigen-binding
specificity to the antibody. However, even a single variable domain (or
half of an Fv comprising only three CDRs specific for an antigen) has the
ability to recognize and bind antigen, although at a lower affinity than
the entire binding site.

[0153]The Fab fragment also contains the constant domain of the light
chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few residues at
the carboxy terminus of the heavy chain CH1 domain including one or more
cysteines from the antibody hinge region. Fab'-SH is the designation
herein for Fab' in which the cysteine residue(s) of the constant domains
bear a free thiol group. F(ab')2 antibody fragments originally were
produced as pairs of Fab' fragments which have hinge cysteines between
them. Other chemical couplings of antibody fragments are also known.

[0154]The "light chains" of antibodies (immunoglobulins) from any
vertebrate species can be assigned to one of two clearly distinct types,
called kappa (κ) and lambda (λ), based on the amino acid
sequences of their constant domains.

[0155]Depending on the amino acid sequence of the constant domain of their
heavy chains, immunoglobulins can be assigned to different classes. There
are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM,
and several of these can be further divided into subclasses (isotypes),
e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and
IgA2. The heavy-chain constant domains that correspond to the
different classes of immunoglobulins are called α, δ,
ε, γ, and μ, respectively. The subunit structures and
three-dimensional configurations of different classes of immunoglobulins
are well known.

[0156]"Antibody fragments" comprise only a portion of an intact antibody,
wherein the portion preferably retains at least one, preferably most or
all, of the functions normally associated with that portion when present
in an intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain
antibody molecules; and multispecific antibodies formed from antibody
fragments. In one embodiment, an antibody fragment comprises an antigen
binding site of the intact antibody and thus retains the ability to bind
antigen. In another embodiment, an antibody fragment, for example one
that comprises the Fc region, retains at least one of the biological
functions normally associated with the Fc region when present in an
intact antibody, such as FcRn binding, antibody half life modulation,
ADCC function and complement binding. In one embodiment, an antibody
fragment is a monovalent antibody that has an in vivo half life
substantially similar to an intact antibody. For e.g., such an antibody
fragment may comprise on antigen binding arm linked to an Fc sequence
capable of conferring in vivo stability to the fragment.

[0157]The term "hypervariable region", "HVR", or "HV", when used herein
refers to the regions of an antibody variable domain which are
hypervariable in sequence and/or form structurally defined loops.
Generally, antibodies comprise six hypervariable regions; three in the VH
(H1, H2, H3), and three in the VL (L1, L2, L3). A number of hypervariable
region delineations are in use and are encompassed herein. The Kabat
Complementarity Determining Regions (CDRs) are based on sequence
variability and are the most commonly used (Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)). Chothia refers
instead to the location of the structural loops (Chothia and Lesk J. Mol.
Biol. 196:901-917 (1987)). The AbM hypervariable regions represent a
compromise between the Kabat CDRs and Chothia structural loops, and are
used by Oxford Molecular's AbM antibody modeling software. The "contact"
hypervariable regions are based on an analysis of the available complex
crystal structures. The residues from each of these hypervariable regions
are noted below.

[0158]Hypervariable regions may comprise "extended hypervariable regions"
as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in
the VL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102
(H3) in the VH. The variable domain residues are numbered according to
Kabat et al., supra for each of these definitions.

[0159]"Framework" or "FR" residues are those variable domain residues
other than the hypervariable region residues as herein defined.

[0160]The term "monoclonal antibody" as used herein refers to an antibody
from a population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical and/or bind
the same epitope(s), except for possible variants that may arise during
production of the monoclonal antibody, such variants generally being
present in minor amounts. Such monoclonal antibody typically includes an
antibody comprising a polypeptide sequence that binds a target, wherein
the target-binding polypeptide sequence was obtained by a process that
includes the selection of a single target binding polypeptide sequence
from a plurality of polypeptide sequences. For example, the selection
process can be the selection of a unique clone from a plurality of
clones, such as a pool of hybridoma clones, phage clones or recombinant
DNA clones. It should be understood that the selected target binding
sequence can be further altered, for example, to improve affinity for the
target, to humanize the target binding sequence, to improve its
production in cell culture, to reduce its immunogenicity in vivo, to
create a multispecific antibody, etc., and that an antibody comprising
the altered target binding sequence is also a monoclonal antibody of this
invention. In contrast to polyclonal antibody preparations which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. In addition to their specificity, the monoclonal antibody
preparations are advantageous in that they are typically uncontaminated
by other immunoglobulins. The modifier "monoclonal" indicates the
character of the antibody as being obtained from a substantially
homogeneous population of antibodies, and is not to be construed as
requiring production of the antibody by any particular method. For
example, the monoclonal antibodies to be used in accordance with the
present invention may be made by a variety of techniques, including, for
example, the hybridoma method (e.g., Kohler et al., Nature, 256:495
(1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring
Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681, (Elsevier, N.Y., 1981)),
recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567), phage
display technologies (see, e.g., Clackson et al., Nature, 352:624-628
(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J.
Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol.
340(5):1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA
101(34):12467-12472 (2004); and Lee et al. J. Immunol. Methods
284(1-2):119-132 (2004), and technologies for producing human or
human-like antibodies in animals that have parts or all of the human
immunoglobulin loci or genes encoding human immunoglobulin sequences
(see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741;
Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits
et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno.,
7:33 (1993); U.S. Pat. Nos. 5,545,806; 5,569,825; 5,591,669 (all of
GenPharm); 5,545,807; WO 1997/17852; U.S. Pat. Nos. 5,545,807; 5,545,806;
5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al.,
Bio/Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859
(1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature
Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14:
826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93
(1995).

[0161]"Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from a
hypervariable region of a non-human species (donor antibody) such as
mouse, rat, rabbit or nonhuman primate having the desired specificity,
affinity, and capacity. In some instances, framework region (FR) residues
of the human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, humanized antibodies may comprise residues that
are not found in the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of a
non-human immunoglobulin and all or substantially all of the FRs are
those of a human immunoglobulin sequence. The humanized antibody
optionally will also comprise at least a portion of an immunoglobulin
constant region (Fc), typically that of a human immunoglobulin. For
further details, see Jones et al., Nature 321:522-525 (1986); Riechmann
et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992). See also the following review articles and references
cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol.
1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995);
Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0162]"Chimeric" antibodies (immunoglobulins) have a portion of the heavy
and/or light chain identical with or homologous to corresponding
sequences in antibodies derived from a particular species or belonging to
a particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences in
antibodies derived from another species or belonging to another antibody
class or subclass, as well as fragments of such antibodies, so long as
they exhibit the desired biological activity (U.S. Pat. No. 4,816,567;
and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
Humanized antibody as used herein is a subset of chimeric antibodies.

[0163]"Single-chain Fv" or "scFv" antibody fragments comprise the VH and
VL domains of antibody, wherein these domains are present in a single
polypeptide chain. Generally, the scFv polypeptide further comprises a
polypeptide linker between the VH and VL domains which enables the scFv
to form the desired structure for antigen binding. For a review of scFv
see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0164]An "antigen" is a predetermined antigen to which an antibody can
selectively bind. The target antigen may be polypeptide, carbohydrate,
nucleic acid, lipid, hapten or other naturally occurring or synthetic
compound. Preferably, the target antigen is a polypeptide.

[0165]The term "diabodies" refers to small antibody fragments with two
antigen-binding sites, which fragments comprise a heavy-chain variable
domain (VH) connected to a light-chain variable domain (VL) in the same
polypeptide chain (VH-VL). By using a linker that is too short to allow
pairing between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create two
antigen-binding sites. Diabodies are described more fully in, for
example, EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad.
Sci. USA, 90:6444-6448 (1993).

[0166]A "human antibody" is one which possesses an amino acid sequence
which corresponds to that of an antibody produced by a human and/or has
been made using any of the techniques for making human antibodies as
disclosed herein. This definition of a human antibody specifically
excludes a humanized antibody comprising non-human antigen-binding
residues.

[0167]An "affinity matured" antibody is one with one or more alterations
in one or more CDRs thereof which result in an improvement in the
affinity of the antibody for antigen, compared to a parent antibody which
does not possess those alteration(s). Preferred affinity matured
antibodies will have nanomolar or even picomolar affinities for the
target antigen. Affinity matured antibodies are produced by procedures
known in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes
affinity maturation by VH and VL domain shuffling. Random mutagenesis of
CDR and/or framework residues is described by: Barbas et al. Proc Nat.
Acad. Sci. USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.,
J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.
226:889-896 (1992).

[0169]"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to
a form of cytotoxicity in which secreted Ig bound onto Fc receptors
(FcRs) present on certain cytotoxic cells (e.g. Natural Killer (NK)
cells, neutrophils, and macrophages) enable these cytotoxic effector
cells to bind specifically to an antigen-bearing target cell and
subsequently kill the target cell with cytotoxins. The antibodies "arm"
the cytotoxic cells and are absolutely required for such killing. The
primary cells for mediating ADCC, NK cells, express FcγRIII only,
whereas monocytes express FcγRI, FcγRII and
FcγRIII. FcR expression on hematopoietic cells is summarized in
Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92
(1991). To assess ADCC activity of a molecule of interest, an in vitro
ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or
5,821,337 or Presta U.S. Pat. No. 6,737,056 may be performed. Useful
effector cells for such assays include peripheral blood mononuclear cells
(PBMC) and Natural Killer (NK) cells. Alternatively, or additionally,
ADCC activity of the molecule of interest may be assessed in vivo, e.g.,
in a animal model such as that disclosed in Clynes et al. Proc. Natl.
Acad. Sci. USA 95:652-656 (1998).

[0170]"Human effector cells" are leukocytes which express one or more FcRs
and perform effector functions. Preferably, the cells express at least
FcγRIII and perform ADCC effector function. Examples of human
leukocytes which mediate ADCC include peripheral blood mononuclear cells
(PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and
neutrophils; with PBMCs and NK cells being preferred. The effector cells
may be isolated from a native source, e.g. from blood.

[0171]"Fc receptor" or "FcR" describes a receptor that binds to the Fc
region of an antibody. The preferred FcR is a native sequence human FcR.
Moreover, a preferred FcR is one which binds an IgG antibody (a gamma
receptor) and includes receptors of the FcγRI, FcγRII,
and FcγRIII subclasses, including allelic variants and
alternatively spliced forms of these receptors. FcγRII receptors
include FcγRIIA (an "activating receptor") and FcγRIIB (an
"inhibiting receptor"), which have similar amino acid sequences that
differ primarily in the cytoplasmic domains thereof. Activating receptor
FcγRIIA contains an immunoreceptor tyrosine-based activation motif
(ITAM) in its cytoplasmic domain Inhibiting receptor FcγRIIB
contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its
cytoplasmic domain. (see review M. in Daeron, Annu. Rev. Immunol.
15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev.
Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and
de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs,
including those to be identified in the future, are encompassed by the
term "FcR" herein. The term also includes the neonatal receptor, FcRn,
which is responsible for the transfer of maternal IgGs to the fetus
(Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol.
24:249 (1994)) and regulates homeostasis of immunoglobulins. WO00/42072
(Presta) describes antibody variants with improved or diminished binding
to FcRs. The content of that patent publication is specifically
incorporated herein by reference. See, also, Shields et al. J. Biol.
Chem. 9(2): 6591-6604 (2001).

[0172]Methods of measuring binding to FcRn are known (see, e.g., Ghetie
1997, Hinton 2004). Binding to human FcRn in vivo and serum half life of
human FcRn high affinity binding polypeptides can be assayed, e.g., in
transgenic mice or transfected human cell lines expressing human FcRn, or
in primates administered with the Fc variant polypeptides.

[0173]"Complement dependent cytotoxicity" or "CDC" refers to the lysis of
a target cell in the presence of complement. Activation of the classical
complement pathway is initiated by the binding of the first component of
the complement system (C1 q) to antibodies (of the appropriate subclass)
which are bound to their cognate antigen. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996), may be performed.

[0175]The term "Fc region-comprising polypeptide" refers to a polypeptide,
such as an antibody or immunoadhesin (see definitions below), which
comprises an Fc region. The C-terminal lysine (residue 447 according to
the EU numbering system) of the Fc region may be removed, for example,
during purification of the polypeptide or by recombinant engineering the
nucleic acid encoding the polypeptide. Accordingly, a composition
comprising a polypeptide having an Fc region according to this invention
can comprise polypeptides with K447, with all K447 removed, or a mixture
of polypeptides with and without the K447 residue.

[0176]A "blocking" antibody or an "antagonist" antibody is one which
inhibits or reduces biological activity of the antigen it binds.
Preferred blocking antibodies or antagonist antibodies substantially or
completely inhibit the biological activity of the antigen.

[0177]An "agonist antibody", as used herein, is an antibody which mimics
at least one of the functional activities of a polypeptide of interest.

[0178]An "acceptor human framework" for the purposes herein is a framework
comprising the amino acid sequence of a VL or VH framework derived from a
human immunoglobulin framework, or from a human consensus framework. An
acceptor human framework "derived from" a human immunoglobulin framework
or human consensus framework may comprise the same amino acid sequence
thereof, or may contain pre-existing amino acid sequence changes. Where
pre-existing amino acid changes are present, preferably no more than 5
and preferably 4 or less, or 3 or less, pre-existing amino acid changes
are present. Where pre-existing amino acid changes are present in a VH,
preferably those changes are only at three, two or one of positions 71H,
73H and 78H; for instance, the amino acid residues at those positions may
be 71A, 73T and/or 78A. In one embodiment, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.

[0179]A "human consensus framework" is a framework which represents the
most commonly occurring amino acid residue in a selection of human
immunoglobulin VL or VH framework sequences. Generally, the selection of
human immunoglobulin VL or VH sequences is from a subgroup of variable
domain sequences. Generally, the subgroup of sequences is a subgroup as
in Kabat et al. In one embodiment, for the VL, the subgroup is subgroup
kappa I as in Kabat et al. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al.

[0180]A "VH subgroup III consensus framework" comprises the consensus
sequence obtained from the amino acid sequences in variable heavy
subgroup III of Kabat et al. In one embodiment, the VH subgroup III
consensus framework amino acid sequence comprises at least a portion or
all of each of the following sequences:

[0181]A "VL subgroup I consensus framework" comprises the consensus
sequence obtained from the amino acid sequences in variable light kappa
subgroup I of Kabat et al. In one embodiment, the VH subgroup I consensus
framework amino acid sequence comprises at least a portion or all of each
of the following sequences:

[0182]A "disorder" or "disease" is any condition that would benefit from
treatment with a substance/molecule or method of the invention. This
includes chronic and acute disorders or diseases including those
pathological conditions which predispose the mammal to the disorder in
question. Non-limiting examples of disorders to be treated herein include
malignant and benign tumors; carcinoma, blastoma, and sarcoma.

[0183]The terms "cell proliferative disorder" and "proliferative disorder"
refer to disorders that are associated with some degree of abnormal cell
proliferation. In one embodiment, the cell proliferative disorder is
cancer.

[0184]"Tumor", as used herein, refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous and
cancerous cells and tissues. The terms "cancer", "cancerous", "cell
proliferative disorder", "proliferative disorder" and "tumor" are not
mutually exclusive as referred to herein.

[0186]As used herein, "treatment" refers to clinical intervention in an
attempt to alter the natural course of the individual or cell being
treated, and can be performed either for prophylaxis or during the course
of clinical pathology. Desirable effects of treatment include preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of the
disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of the
invention are used to delay development of a disease or disorder.

[0187]An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to, farm animals
(such as cows), sport animals, pets (such as cats, dogs and horses),
primates, mice and rats.

[0188]"Mammal" for purposes of treatment refers to any animal classified
as a mammal, including humans, domestic and farm animals, and zoo,
sports, or pet animals, such as dogs, horses, cats, cows, etc.
Preferably, the mammal is human.

[0189]An "effective amount" refers to an amount effective, at dosages and
for periods of time necessary, to achieve the desired therapeutic or
prophylactic result.

[0190]A "therapeutically effective amount" of a substance/molecule of the
invention, agonist or antagonist may vary according to factors such as
the disease state, age, sex, and weight of the individual, and the
ability of the substance/molecule, agonist or antagonist to elicit a
desired response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the
substance/molecule, agonist or antagonist are outweighed by the
therapeutically beneficial effects. A "prophylactically effective amount"
refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired prophylactic result. Typically but not
necessarily, since a prophylactic dose is used in subjects prior to or at
an earlier stage of disease, the prophylactically effective amount will
be less than the therapeutically effective amount.

[0191]The term "cytotoxic agent" as used herein refers to a substance that
inhibits or prevents the function of cells and/or causes destruction of
cells. The term is intended to include radioactive isotopes (e.g.,
At211, I131, I125, Y90, Re186, Re188,
Sm153, Bi212, P32 and radioactive isotopes of Lu),
chemotherapeutic agents e.g. methotrexate, adriamicin, vinca alkaloids
(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin
C, chlorambucil, daunorubicin or other intercalating agents, enzymes and
fragments thereof such as nucleolytic enzymes, antibiotics, and toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments and/or
variants thereof, and the various antitumor or anticancer agents
disclosed below. Other cytotoxic agents are described below. A
tumoricidal agent causes destruction of tumor cells.

[0193]Also included in this definition are anti-hormonal agents that act
to regulate, reduce, block, or inhibit the effects of hormones that can
promote the growth of cancer, and are often in the form of systemic, or
whole-body treatment. They may be hormones themselves. Examples include
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX® tamoxifen),
EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,
keoxifene, LY117018, onapristone, and FARESTON® toremifene;
anti-progesterones; estrogen receptor down-regulators (ERDs); agents that
function to suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON® and
ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetate and
tripterelin; other anti-androgens such as flutamide, nilutamide and
bicalutamide; and aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as, for
example, 4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol
acetate, AROMASIN® exemestane, formestanie, fadrozole, RIVISOR®
vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. In
addition, such definition of chemotherapeutic agents includes
bisphosphonates such as clodronate (for example, BONEFOS® or
OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic
acid/zoledronate, FOSAMAX® alendronate, AREDIA® pamidronate,
SKELID® tiludronate, or ACTONEL® risedronate; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense
oligonucleotides, particularly those that inhibit expression of genes in
signaling pathways implicated in abherant cell proliferation, such as,
for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor
(EGF-R); vaccines such as THERATOPE® vaccine and gene therapy
vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine,
and VAXID® vaccine; LURTOTECAN® topoisomerase 1 inhibitor;
ABARELIX® rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual
tyrosine kinase small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of the
above.

[0194]A "growth inhibitory agent" when used herein refers to a compound or
composition which inhibits growth of a cell (such as a cell expressing
DLL4) either in vitro or in vivo. Thus, the growth inhibitory agent may
be one which significantly reduces the percentage of cells (such as a
cell expressing DLL4) in S phase. Examples of growth inhibitory agents
include agents that block cell cycle progression (at a place other than S
phase), such as agents that induce G1 arrest and M-phase arrest.
Classical M-phase blockers include the vincas (vincristine and
vinblastine), taxanes, and topoisomerase II inhibitors such as
doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those
agents that arrest G1 also spill over into S-phase arrest, for example,
DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,
mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
Further information can be found in The Molecular Basis of Cancer,
Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation,
oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and
docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel
(TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a
semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb).
Paclitaxel and docetaxel promote the assembly of microtubules from
tubulin dimers and stabilize microtubules by preventing depolymerization,
which results in the inhibition of mitosis in cells.

[0195]"Doxorubicin" is an anthracycline antibiotic. The full chemical name
of doxorubicin is
(85-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.

[0197]The term "prodrug" as used in this application refers to a precursor
or derivative form of a pharmaceutically active substance that is less
cytotoxic to tumor cells compared to the parent drug and is capable of
being enzymatically activated or converted into the more active parent
form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical
Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and
Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery,"
Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana
Press (1985). The prodrugs of this invention include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs, glycosylated prodrugs, beta-lactam-containing
prodrugs, optionally substituted phenoxyacetamide-containing prodrugs or
optionally substituted phenylacetamide-containing prodrugs,
5-fluorocytosine and other 5-fluorouridine prodrugs which can be
converted into the more active cytotoxic free drug. Examples of cytotoxic
drugs that can be derivatized into a prodrug form for use in this
invention include, but are not limited to, those chemotherapeutic agents
described above.

[0199]This invention encompasses compositions, including pharmaceutical
compositions, comprising an anti-DLL4 antibody; and polynucleotides
comprising sequences encoding an anti-DLL4 antibody. As used herein,
compositions comprise one or more antibodies that bind to DLL4, and/or
one or more polynucleotides comprising sequences encoding one or more
antibodies that bind to DLL4. These compositions may further comprise
suitable carriers, such as pharmaceutically acceptable excipients
including buffers, which are well known in the art.

[0201]The anti-DLL4 antibodies of the invention are preferably monoclonal.
Also encompassed within the scope of the invention are Fab, Fab', Fab'-SH
and F(ab')2 fragments of the anti-DLL4 antibodies provided herein.
These antibody fragments can be created by traditional means, such as
enzymatic digestion, or may be generated by recombinant techniques. Such
antibody fragments may be chimeric or humanized. These fragments are
useful for the diagnostic and therapeutic purposes set forth below.

[0202]Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are identical except for possible naturally
occurring mutations that may be present in minor amounts. Thus, the
modifier "monoclonal" indicates the character of the antibody as not
being a mixture of discrete antibodies.

[0203]The anti-DLL4 monoclonal antibodies of the invention can be made
using the hybridoma method first described by Kohler et al., Nature,
256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No.
4,816,567).

[0204]In the hybridoma method, a mouse or other appropriate host animal,
such as a hamster, is immunized to elicit lymphocytes that produce or are
capable of producing antibodies that will specifically bind to the
protein used for immunization. Antibodies to DLL4 generally are raised in
animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections
of DLL4 and an adjuvant. DLL4 may be prepared using methods well-known in
the art, some of which are further described herein. For example,
recombinant production of DLL4 is described below. In one embodiment,
animals are immunized with a derivative of DLL4 that contains the
extracellular domain (ECD) of DLL4 fused to the Fc portion of an
immunoglobulin heavy chain. In a preferred embodiment, animals are
immunized with an DLL4-IgG1 fusion protein. Animals ordinarily are
immunized against immunogenic conjugates or derivatives of DLL4 with
monophosphoryl lipid A (MPL)/trehalose dicrynomycolate (TDM) (Bibi
Immunochem. Research, Inc., Hamilton, Mont.) and the solution is injected
intradermally at multiple sites. Two weeks later the animals are boosted.
7 to 14 days later animals are bled and the serum is assayed for
anti-DLL4 titer. Animals are boosted until titer plateaus.

[0205]Alternatively, lymphocytes may be immunized in vitro. Lymphocytes
then are fused with myeloma cells using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0206]The hybridoma cells thus prepared are seeded and grown in a suitable
culture medium that preferably contains one or more substances that
inhibit the growth or survival of the unfused, parental myeloma cells.
For example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium
for the hybridomas typically will include hypoxanthine, aminopterin, and
thymidine (HAT medium), which substances prevent the growth of
HGPRT-deficient cells.

[0207]Preferred myeloma cells are those that fuse efficiently, support
stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine myeloma
lines, such as those derived from MOPC-21 and MPC-11 mouse tumors
available from the Salk Institute Cell Distribution Center, San Diego,
Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American
Type Culture Collection, Rockville, Md. USA. Human myeloma and
mouse-human heteromyeloma cell lines also have been described for the
production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001
(1984); Brodeur et al., Monoclonal Antibody Production Techniques and
Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0208]Culture medium in which hybridoma cells are growing is assayed for
production of monoclonal antibodies directed against DLL4. Preferably,
the binding specificity of monoclonal antibodies produced by hybridoma
cells is determined by immunoprecipitation or by an in vitro binding
assay, such as radioimmunoassay (RIA) or enzyme-linked immunoadsorbent
assay (ELISA).

[0210]After hybridoma cells are identified that produce antibodies of the
desired specificity, affinity, and/or activity, the clones may be
subcloned by limiting dilution procedures and grown by standard methods
(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103
(Academic Press, 1986)). Suitable culture media for this purpose include,
for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells
may be grown in vivo as ascites tumors in an animal.

[0212]The anti-DLL4 antibodies of the invention can be made by using
combinatorial libraries to screen for synthetic antibody clones with the
desired activity or activities. In principle, synthetic antibody clones
are selected by screening phage libraries containing phage that display
various fragments of antibody variable region (Fv) fused to phage coat
protein. Such phage libraries are panned by affinity chromatography
against the desired antigen. Clones expressing Fv fragments capable of
binding to the desired antigen are adsorbed to the antigen and thus
separated from the non-binding clones in the library. The binding clones
are then eluted from the antigen, and can be further enriched by
additional cycles of antigen adsorption/elution. Any of the anti-DLL4
antibodies of the invention can be obtained by designing a suitable
antigen screening procedure to select for the phage clone of interest
followed by construction of a full length anti-DLL4 antibody clone using
the Fv sequences from the phage clone of interest and suitable constant
region (Fc) sequences described in Kabat et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda
Md. (1991), vols. 1-3.

[0213]The antigen-binding domain of an antibody is formed from two
variable (V) regions of about 110 amino acids, one each from the light
(VL) and heavy (VH) chains, that both present three hypervariable loops
or complementarity-determining regions (CDRs). Variable domains can be
displayed functionally on phage, either as single-chain Fv (scFv)
fragments, in which VH and VL are covalently linked through a short,
flexible peptide, or as Fab fragments, in which they are each fused to a
constant domain and interact non-covalently, as described in Winter et
al., Ann. Rev. Immunol., 12: 433-455 (1994). As used herein, scFv
encoding phage clones and Fab encoding phage clones are collectively
referred to as "Fv phage clones" or "Fv clones".

[0214]Repertoires of VH and VL genes can be separately cloned by
polymerase chain reaction (PCR) and recombined randomly in phage
libraries, which can then be searched for antigen-binding clones as
described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
Libraries from immunized sources provide high-affinity antibodies to the
immunogen without the requirement of constructing hybridomas.
Alternatively, the naive repertoire can be cloned to provide a single
source of human antibodies to a wide range of non-self and also self
antigens without any immunization as described by Griffiths et al., EMBO
J, 12: 725-734 (1993). Finally, naive libraries can also be made
synthetically by cloning the unrearranged V-gene segments from stem
cells, and using PCR primers containing random sequence to encode the
highly variable CDR3 regions and to accomplish rearrangement in vitro as
described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).

[0215]Filamentous phage is used to display antibody fragments by fusion to
the minor coat protein pIII. The antibody fragments can be displayed as
single chain Fv fragments, in which VH and VL domains are connected on
the same polypeptide chain by a flexible polypeptide spacer, e.g. as
described by Marks et al., J. Mol. Biol., 222: 581-597 (1991), or as Fab
fragments, in which one chain is fused to pIII and the other is secreted
into the bacterial host cell periplasm where assembly of a Fab-coat
protein structure which becomes displayed on the phage surface by
displacing some of the wild type coat proteins, e.g. as described in
Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991).

[0216]In general, nucleic acids encoding antibody gene fragments are
obtained from immune cells harvested from humans or animals. If a library
biased in favor of anti-DLL4 clones is desired, the subject is immunized
with DLL4 to generate an antibody response, and spleen cells and/or
circulating B cells other peripheral blood lymphocytes (PBLs) are
recovered for library construction. In a preferred embodiment, a human
antibody gene fragment library biased in favor of anti-DLL4 clones is
obtained by generating an anti-DLL4 antibody response in transgenic mice
carrying a functional human immunoglobulin gene array (and lacking a
functional endogenous antibody production system) such that DLL4
immunization gives rise to B cells producing human antibodies against
DLL4. The generation of human antibody-producing transgenic mice is
described below.

[0217]Additional enrichment for anti-DLL4 reactive cell populations can be
obtained by using a suitable screening procedure to isolate B cells
expressing DLL4-specific membrane bound antibody, e.g., by cell
separation with DLL4 affinity chromatography or adsorption of cells to
fluorochrome-labeled DLL4 followed by flow-activated cell sorting (FACS).

[0218]Alternatively, the use of spleen cells and/or B cells or other PBLs
from an unimmunized donor provides a better representation of the
possible antibody repertoire, and also permits the construction of an
antibody library using any animal (human or non-human) species in which
DLL4 is not antigenic. For libraries incorporating in vitro antibody gene
construction, stem cells are harvested from the subject to provide
nucleic acids encoding unrearranged antibody gene segments. The immune
cells of interest can be obtained from a variety of animal species, such
as human, mouse, rat, lagomorpha, luprine, canine, feline, porcine,
bovine, equine, and avian species, etc.

[0219]Nucleic acid encoding antibody variable gene segments (including VH
and VL segments) are recovered from the cells of interest and amplified.
In the case of rearranged VH and VL gene libraries, the desired DNA can
be obtained by isolating genomic DNA or mRNA from lymphocytes followed by
polymerase chain reaction (PCR) with primers matching the 5' and 3' ends
of rearranged VH and VL genes as described in Orlandi et al., Proc. Natl.
Acad. Sci. (USA), 86: 3833-3837 (1989), thereby making diverse V gene
repertoires for expression. The V genes can be amplified from cDNA and
genomic DNA, with back primers at the 5' end of the exon encoding the
mature V-domain and forward primers based within the J-segment as
described in Orlandi et al. (1989) and in Ward et al., Nature, 341:
544-546 (1989). However, for amplifying from cDNA, back primers can also
be based in the leader exon as described in Jones et al., Biotechnol., 9:
88-89 (1991), and forward primers within the constant region as described
in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86: 5728-5732 (1989). To
maximize complementarity, degeneracy can be incorporated in the primers
as described in Orlandi et al. (1989) or Sastry et al. (1989).
Preferably, the library diversity is maximized by using PCR primers
targeted to each V-gene family in order to amplify all available VH and
VL arrangements present in the immune cell nucleic acid sample, e.g. as
described in the method of Marks et al., J. Mol. Biol., 222: 581-597
(1991) or as described in the method of Orum et al., Nucleic Acids Res.,
21: 4491-4498 (1993). For cloning of the amplified DNA into expression
vectors, rare restriction sites can be introduced within the PCR primer
as a tag at one end as described in Orlandi et al. (1989), or by further
PCR amplification with a tagged primer as described in Clackson et al.,
Nature, 352: 624-628 (1991).

[0220]Repertoires of synthetically rearranged V genes can be derived in
vitro from V gene segments. Most of the human VH-gene segments have been
cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227:
776-798 (1992)), and mapped (reported in Matsuda et al., Nature Genet.,
3: 88-94 (1993); these cloned segments (including all the major
conformations of the H1 and H2 loop) can be used to generate diverse VH
gene repertoires with PCR primers encoding H3 loops of diverse sequence
and length as described in Hoogenboom and Winter, J. Mol. Biol., 227:
381-388 (1992). VH repertoires can also be made with all the sequence
diversity focused in a long H3 loop of a single length as described in
Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-4461 (1992). Human
Vκ and Vλ segments have been cloned and sequenced (reported
in Williams and Winter, Eur. J. Immunol., 23: 1456-1461 (1993)) and can
be used to make synthetic light chain repertoires. Synthetic V gene
repertoires, based on a range of VH and VL folds, and L3 and H3 lengths,
will encode antibodies of considerable structural diversity. Following
amplification of V-gene encoding DNAs, germline V-gene segments can be
rearranged in vitro according to the methods of Hoogenboom and Winter, J.
Mol. Biol., 227: 381-388 (1992).

[0221]Repertoires of antibody fragments can be constructed by combining VH
and VL gene repertoires together in several ways. Each repertoire can be
created in different vectors, and the vectors recombined in vitro, e.g.,
as described in Hogrefe et al., Gene, 128: 119-126 (1993), or in vivo by
combinatorial infection, e.g., the loxP system described in Waterhouse et
al., Nuci. Acids Res., 21: 2265-2266 (1993). The in vivo recombination
approach exploits the two-chain nature of Fab fragments to overcome the
limit on library size imposed by E. coli transformation efficiency. Naive
VH and VL repertoires are cloned separately, one into a phagemid and the
other into a phage vector. The two libraries are then combined by phage
infection of phagemid-containing bacteria so that each cell contains a
different combination and the library size is limited only by the number
of cells present (about 1012 clones). Both vectors contain in vivo
recombination signals so that the VH and VL genes are recombined onto a
single replicon and are co-packaged into phage virions. These huge
libraries provide large numbers of diverse antibodies of good affinity
(Kd-1 of about 10-8 M).

[0222]Alternatively, the repertoires may be cloned sequentially into the
same vector, e.g. as described in Barbas et al., Proc. Natl. Acad. Sci.
USA, 88: 7978-7982 (1991), or assembled together by PCR and then cloned,
e.g. as described in Clackson et al., Nature, 352: 624-628 (1991). PCR
assembly can also be used to join VH and VL DNAs with DNA encoding a
flexible peptide spacer to form single chain Fv (scFv) repertoires. In
yet another technique, "in cell PCR assembly" is used to combine VH and
VL genes within lymphocytes by PCR and then clone repertoires of linked
genes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837
(1992).

[0223]The antibodies produced by naive libraries (either natural or
synthetic) can be of moderate affinity (Kd-1 of about 106
to 107 M-1), but affinity maturation can also be mimicked in
vitro by constructing and reselecting from secondary libraries as
described in Winter et al. (1994), supra. For example, mutation can be
introduced at random in vitro by using error-prone polymerase (reported
in Leung et al., Technique, 1: 11-15 (1989)) in the method of Hawkins et
al., J. Mol. Biol., 226: 889-896 (1992) or in the method of Gram et al.,
Proc. Natl. Acad. Sci USA, 89: 3576-3580 (1992). Additionally, affinity
maturation can be performed by randomly mutating one or more CDRs, e.g.
using PCR with primers carrying random sequence spanning the CDR of
interest, in selected individual Fv clones and screening for higher
affinity clones. WO 9607754 (published 14 Mar. 1996) described a method
for inducing mutagenesis in a complementarity determining region of an
immunoglobulin light chain to create a library of light chain genes.
Another effective approach is to recombine the VH or VL domains selected
by phage display with repertoires of naturally occurring V domain
variants obtained from unimmunized donors and screen for higher affinity
in several rounds of chain reshuffling as described in Marks et al.,
Biotechnol., 10: 779-783 (1992). This technique allows the production of
antibodies and antibody fragments with affinities in the 10-9 M
range.

[0225]DNAs encoding DLL4 can be prepared by a variety of methods known in
the art. These methods include, but are not limited to, chemical
synthesis by any of the methods described in Engels et al., Agnew. Chem.
Int. Ed. Engl., 28: 716-734 (1989), such as the triester, phosphite,
phosphoramidite and H-phosphonate methods. In one embodiment, codons
preferred by the expression host cell are used in the design of the DLL4
encoding DNA. Alternatively, DNA encoding the DLL4 can be isolated from a
genomic or cDNA library.

[0226]Following construction of the DNA molecule encoding the DLL4, the
DNA molecule is operably linked to an expression control sequence in an
expression vector, such as a plasmid, wherein the control sequence is
recognized by a host cell transformed with the vector. In general,
plasmid vectors contain replication and control sequences which are
derived from species compatible with the host cell. The vector ordinarily
carries a replication site, as well as sequences which encode proteins
that are capable of providing phenotypic selection in transformed cells.
Suitable vectors for expression in prokaryotic and eukaryotic host cells
are known in the art and some are further described herein. Eukaryotic
organisms, such as yeasts, or cells derived from multicellular organisms,
such as mammals, may be used.

[0227]Optionally, the DNA encoding the DLL4 is operably linked to a
secretory leader sequence resulting in secretion of the expression
product by the host cell into the culture medium. Examples of secretory
leader sequences include stII, ecotin, lamB, herpes GD, 1 pp, alkaline
phosphatase, invertase, and alpha factor. Also suitable for use herein is
the 36 amino acid leader sequence of protein A (Abrahmsen et al., EMBO
J., 4: 3901 (1985)).

[0228]Host cells are transfected and preferably transformed with the
above-described expression or cloning vectors of this invention and
cultured in conventional nutrient media modified as appropriate for
inducing promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.

[0229]Transfection refers to the taking up of an expression vector by a
host cell whether or not any coding sequences are in fact expressed.
Numerous methods of transfection are known to the ordinarily skilled
artisan, for example, CaPO4 precipitation and electroporation.
Successful transfection is generally recognized when any indication of
the operation of this vector occurs within the host cell. Methods for
transfection are well known in the art, and some are further described
herein.

[0230]Transformation means introducing DNA into an organism so that the
DNA is replicable, either as an extrachromosomal element or by
chromosomal integrant. Depending on the host cell used, transformation is
done using standard techniques appropriate to such cells. Methods for
transformation are well known in the art, and some are further described
herein.

[0231]Prokaryotic host cells used to produce the DLL4 can be cultured as
described generally in Sambrook et al., supra.

[0232]The mammalian host cells used to produce the DLL4 can be cultured in
a variety of media, which is well known in the art and some of which is
described herein.

[0233]The host cells referred to in this disclosure encompass cells in in
vitro culture as well as cells that are within a host animal.

[0234]Purification of DLL4 may be accomplished using art-recognized
methods, some of which are described herein.

[0235]The purified DLL4 can be attached to a suitable matrix such as
agarose beads, acrylamide beads, glass beads, cellulose, various acrylic
copolymers, hydroxylmethacrylate gels, polyacrylic and polymethacrylic
copolymers, nylon, neutral and ionic carriers, and the like, for use in
the affinity chromatographic separation of phage display clones.
Attachment of the DLL4 protein to the matrix can be accomplished by the
methods described in Methods in Enzymology, vol. 44 (1976). A commonly
employed technique for attaching protein ligands to polysaccharide
matrices, e.g. agarose, dextran or cellulose, involves activation of the
carrier with cyanogen halides and subsequent coupling of the peptide
ligand's primary aliphatic or aromatic amines to the activated matrix.

[0236]Alternatively, DLL4 can be used to coat the wells of adsorption
plates, expressed on host cells affixed to adsorption plates or used in
cell sorting, or conjugated to biotin for capture with
streptavidin-coated beads, or used in any other art-known method for
panning phage display libraries.

[0237]The phage library samples are contacted with immobilized DLL4 under
conditions suitable for binding of at least a portion of the phage
particles with the adsorbent. Normally, the conditions, including pH,
ionic strength, temperature and the like are selected to mimic
physiological conditions. The phages bound to the solid phase are washed
and then eluted by acid, e.g. as described in Barbas et al., Proc. Natl.
Acad. Sci USA, 88: 7978-7982 (1991), or by alkali, e.g. as described in
Marks et al., J. Mol. Biol., 222: 581-597 (1991), or by DLL4 antigen
competition, e.g. in a procedure similar to the antigen competition
method of Clackson et al., Nature, 352: 624-628 (1991). Phages can be
enriched 20-1,000-fold in a single round of selection. Moreover, the
enriched phages can be grown in bacterial culture and subjected to
further rounds of selection.

[0238]The efficiency of selection depends on many factors, including the
kinetics of dissociation during washing, and whether multiple antibody
fragments on a single phage can simultaneously engage with antigen.
Antibodies with fast dissociation kinetics (and weak binding affinities)
can be retained by use of short washes, multivalent phage display and
high coating density of antigen in solid phase. The high density not only
stabilizes the phage through multivalent interactions, but favors
rebinding of phage that has dissociated. The selection of antibodies with
slow dissociation kinetics (and good binding affinities) can be promoted
by use of long washes and monovalent phage display as described in Bass
et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating
density of antigen as described in Marks et al., Biotechnol., 10: 779-783
(1992).

[0239]It is possible to select between phage antibodies of different
affinities, even with affinities that differ slightly, for DLL4. However,
random mutation of a selected antibody (e.g. as performed in some of the
affinity maturation techniques described above) is likely to give rise to
many mutants, most binding to antigen, and a few with higher affinity.
With limiting DLL4, rare high affinity phage could be competed out. To
retain all the higher affinity mutants, phages can be incubated with
excess biotinylated DLL4, but with the biotinylated DLL4 at a
concentration of lower molarity than the target molar affinity constant
for DLL4. The high affinity-binding phages can then be captured by
streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows
the antibodies to be selected according to their affinities of binding,
with sensitivity that permits isolation of mutant clones with as little
as two-fold higher affinity from a great excess of phages with lower
affinity. Conditions used in washing phages bound to a solid phase can
also be manipulated to discriminate on the basis of dissociation
kinetics.

[0240]Anti-DLL4 clones may be activity selected. In one embodiment, the
invention provides anti-DLL4 antibodies that block the binding between a
Notch receptor (such as Notch1, Notch2, Notch3 and/or Notch4) and DLL4,
but do not block the binding between a Notch receptor and a second
protein. Fv clones corresponding to such anti-DLL4 antibodies can be
selected by (1) isolating anti-DLL4 clones from a phage library as
described above, and optionally amplifying the isolated population of
phage clones by growing up the population in a suitable bacterial host;
(2) selecting DLL4 and a second protein against which blocking and
non-blocking activity, respectively, is desired; (3) adsorbing the
anti-DLL4 phage clones to immobilized DLL4; (4) using an excess of the
second protein to elute any undesired clones that recognize DLL4-binding
determinants which overlap or are shared with the binding determinants of
the second protein; and (5) eluting the clones which remain adsorbed
following step (4). Optionally, clones with the desired
blocking/non-blocking properties can be further enriched by repeating the
selection procedures described herein one or more times.

[0241]DNA encoding the hybridoma-derived monoclonal antibodies or phage
display Fv clones of the invention is readily isolated and sequenced
using conventional procedures (e.g. by using oligonucleotide primers
designed to specifically amplify the heavy and light chain coding regions
of interest from hybridoma or phage DNA template). Once isolated, the DNA
can be placed into expression vectors, which are then transfected into
host cells such as E. coli cells, simian COS cells, Chinese hamster ovary
(CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of the desired monoclonal
antibodies in the recombinant host cells. Review articles on recombinant
expression in bacteria of antibody-encoding DNA include Skerra et al.,
Curr. Opinion in Immunol., 5: 256 (1993) and Pluckthun, Immunol. Revs,
130: 151 (1992).

[0242]DNA encoding the Fv clones of the invention can be combined with
known DNA sequences encoding heavy chain and/or light chain constant
regions (e.g. the appropriate DNA sequences can be obtained from Kabat et
al., supra) to form clones encoding full or partial length heavy and/or
light chains. It will be appreciated that constant regions of any isotype
can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE
constant regions, and that such constant regions can be obtained from any
human or animal species. A Fv clone derived from the variable domain DNA
of one animal (such as human) species and then fused to constant region
DNA of another animal species to form coding sequence(s) for "hybrid",
full length heavy chain and/or light chain is included in the definition
of "chimeric" and "hybrid" antibody as used herein. In a preferred
embodiment, a Fv clone derived from human variable DNA is fused to human
constant region DNA to form coding sequence(s) for all human, full or
partial length heavy and/or light chains.

[0243]DNA encoding anti-DLL4 antibody derived from a hybridoma of the
invention can also be modified, for example, by substituting the coding
sequence for human heavy- and light-chain constant domains in place of
homologous murine sequences derived from the hybridoma clone (e.g. as in
the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855
(1984)). DNA encoding a hybridoma or Fv clone-derived antibody or
fragment can be further modified by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide. In this manner, "chimeric" or "hybrid"
antibodies are prepared that have the binding specificity of the Fv clone
or hybridoma clone-derived antibodies of the invention.

Antibody Fragments

[0244]The present invention encompasses antibody fragments. In certain
circumstances there are advantages of using antibody fragments, rather
than whole antibodies. The smaller size of the fragments allows for rapid
clearance, and may lead to improved access to solid tumors.

[0245]Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et al.,
Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and
Brennan et al., Science, 229:81 (1985)). However, these fragments can now
be produced directly by recombinant host cells. Fab, Fv and ScFv antibody
fragments can all be expressed in and secreted from E. coli, thus
allowing the facile production of large amounts of these fragments.
Antibody fragments can be isolated from the antibody phage libraries
discussed above. Alternatively, Fab'-SH fragments can be directly
recovered from E. coli and chemically coupled to form F(ab')2
fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to
another approach, F(ab')2 fragments can be isolated directly from
recombinant host cell culture. Fab and F(ab')2 fragment with
increased in vivo half-life comprising a salvage receptor binding epitope
residues are described in U.S. Pat. No. 5,869,046. Other techniques for
the production of antibody fragments will be apparent to the skilled
practitioner. In other embodiments, the antibody of choice is a single
chain Fv fragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and
5,587,458. Fv and sFv are the only species with intact combining sites
that are devoid of constant regions; thus, they are suitable for reduced
nonspecific binding during in vivo use. sFv fusion proteins may be
constructed to yield fusion of an effector protein at either the amino or
the carboxy terminus of an sFv. See Antibody Engineering, ed. Borrebaeck,
supra. The antibody fragment may also be a "linear antibody", e.g., as
described in U.S. Pat. No. 5,641,870 for example. Such linear antibody
fragments may be monospecific or bispecific.

Humanized Antibodies

[0246]The present invention encompasses humanized antibodies. Various
methods for humanizing non-human antibodies are known in the art. For
example, a humanized antibody can have one or more amino acid residues
introduced into it from a source which is non-human. These non-human
amino acid residues are often referred to as "import" residues, which are
typically taken from an "import" variable domain. Humanization can be
essentially performed following the method of Winter and co-workers
(Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature
332:323-327; Verhoeyen et al. (1988) Science 239:1534-1536), by
substituting hypervariable region sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized" antibodies
are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially
less than an intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some hypervariable
region residues and possibly some FR residues are substituted by residues
from analogous sites in rodent antibodies.

[0247]The choice of human variable domains, both light and heavy, to be
used in making the humanized antibodies is very important to reduce
antigenicity. According to the so-called "best-fit" method, the sequence
of the variable domain of a rodent antibody is screened against the
entire library of known human variable-domain sequences. The human
sequence which is closest to that of the rodent is then accepted as the
human framework for the humanized antibody (Sims et al. (1993) J.
Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Another
method uses a particular framework derived from the consensus sequence of
all human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized antibodies
(Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285; Presta et al.
(1993) J. Immunol., 151:2623.

[0248]It is further important that antibodies be humanized with retention
of high affinity for the antigen and other favorable biological
properties. To achieve this goal, according to one method, humanized
antibodies are prepared by a process of analysis of the parental
sequences and various conceptual humanized products using
three-dimensional models of the parental and humanized sequences.
Three-dimensional immunoglobulin models are commonly available and are
familiar to those skilled in the art. Computer programs are available
which illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences. Inspection of
these displays permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the analysis
of residues that influence the ability of the candidate immunoglobulin to
bind its antigen. In this way, FR residues can be selected and combined
from the recipient and import sequences so that the desired antibody
characteristic, such as increased affinity for the target antigen(s), is
achieved. In general, the hypervariable region residues are directly and
most substantially involved in influencing antigen binding.

Human Antibodies

[0249]Human anti-DLL4 antibodies of the invention can be constructed by
combining Fv clone variable domain sequence(s) selected from
human-derived phage display libraries with known human constant domain
sequences(s) as described above. Alternatively, human monoclonal
anti-DLL4 antibodies of the invention can be made by the hybridoma
method. Human myeloma and mouse-human heteromyeloma cell lines for the
production of human monoclonal antibodies have been described, for
example, by Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp. 51-63
(Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol.,
147: 86 (1991).

[0250]It is now possible to produce transgenic animals (e.g. mice) that
are capable, upon immunization, of producing a full repertoire of human
antibodies in the absence of endogenous immunoglobulin production. For
example, it has been described that the homozygous deletion of the
antibody heavy-chain joining region (JH) gene in chimeric and germ-line
mutant mice results in complete inhibition of endogenous antibody
production. Transfer of the human germ-line immunoglobulin gene array in
such germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc.
Natl. Acad. Sci USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255
(1993); Bruggermann et al., Year in Immunol., 7: 33 (1993).

[0251]Gene shuffling can also be used to derive human antibodies from
non-human, e.g. rodent, antibodies, where the human antibody has similar
affinities and specificities to the starting non-human antibody.
According to this method, which is also called "epitope imprinting",
either the heavy or light chain variable region of a non-human antibody
fragment obtained by phage display techniques as described above is
replaced with a repertoire of human V domain genes, creating a population
of non-human chain/human chain scFv or Fab chimeras. Selection with
antigen results in isolation of a non-human chain/human chain chimeric
scFv or Fab wherein the human chain restores the antigen binding site
destroyed upon removal of the corresponding non-human chain in the
primary phage display clone, i.e. the epitope governs (imprints) the
choice of the human chain partner. When the process is repeated in order
to replace the remaining non-human chain, a human antibody is obtained
(see PCT WO 93/06213 published Apr. 1, 1993). Unlike traditional
humanization of non-human antibodies by CDR grafting, this technique
provides completely human antibodies, which have no FR or CDR residues of
non-human origin.

Bispecific Antibodies

[0252]Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding specificities for at least two different
antigens. In the present case, one of the binding specificities is for
DLL4 and the other is for any other antigen. Exemplary bispecific
antibodies may bind to two different epitopes of the DLL4 protein.
Bispecific antibodies may also be used to localize cytotoxic agents to
cells which express DLL4. These antibodies possess an DLL4-binding arm
and an arm which binds the cytotoxic agent (e.g. saporin,
anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate or
radioactive isotope hapten). Bispecific antibodies can be prepared as
full length antibodies or antibody fragments (e.g. F(ab')2
bispecific antibodies).

[0253]Methods for making bispecific antibodies are known in the art.
Traditionally, the recombinant production of bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light chain
pairs, where the two heavy chains have different specificities (Milstein
and Cuello, Nature, 305: 537 (1983)). Because of the random assortment of
immunoglobulin heavy and light chains, these hybridomas (quadromas)
produce a potential mixture of 10 different antibody molecules, of which
only one has the correct bispecific structure. The purification of the
correct molecule, which is usually done by affinity chromatography steps,
is rather cumbersome, and the product yields are low. Similar procedures
are disclosed in WO 93/08829 published May 13, 1993, and in Traunecker et
al., EMBO J., 10: 3655 (1991).

[0254]According to a different and more preferred approach, antibody
variable domains with the desired binding specificities (antibody-antigen
combining sites) are fused to immunoglobulin constant domain sequences.
The fusion preferably is with an immunoglobulin heavy chain constant
domain, comprising at least part of the hinge, CH2, and CH3 regions. It
is preferred to have the first heavy-chain constant region (CH1),
containing the site necessary for light chain binding, present in at
least one of the fusions. DNAs encoding the immunoglobulin heavy chain
fusions and, if desired, the immunoglobulin light chain, are inserted
into separate expression vectors, and are co-transfected into a suitable
host organism. This provides for great flexibility in adjusting the
mutual proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the coding
sequences for two or all three polypeptide chains in one expression
vector when the expression of at least two polypeptide chains in equal
ratios results in high yields or when the ratios are of no particular
significance.

[0255]In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with a
first binding specificity in one arm, and a hybrid immunoglobulin heavy
chain-light chain pair (providing a second binding specificity) in the
other arm. It was found that this asymmetric structure facilitates the
separation of the desired bispecific compound from unwanted
immunoglobulin chain combinations, as the presence of an immunoglobulin
light chain in only one half of the bispecific molecule provides for a
facile way of separation. This approach is disclosed in WO 94/04690. For
further details of generating bispecific antibodies see, for example,
Suresh et al., Methods in Enzymology, 121:210 (1986).

[0256]According to another approach, the interface between a pair of
antibody molecules can be engineered to maximize the percentage of
heterodimers which are recovered from recombinant cell culture. The
preferred interface comprises at least a part of the CH3 domain of
an antibody constant domain. In this method, one or more small amino acid
side chains from the interface of the first antibody molecule are
replaced with larger side chains (e.g. tyrosine or tryptophan).
Compensatory "cavities" of identical or similar size to the large side
chain(s) are created on the interface of the second antibody molecule by
replacing large amino acid side chains with smaller ones (e.g. alanine or
threonine). This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as homodimers.

[0257]Bispecific antibodies include cross-linked or "heteroconjugate"
antibodies. For example, one of the antibodies in the heteroconjugate can
be coupled to avidin, the other to biotin. Such antibodies have, for
example, been proposed to target immune system cells to unwanted cells
(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO
91/00360, WO 92/00373, and EP 03089). Heteroconjugate antibodies may be
made using any convenient cross-linking methods. Suitable cross-linking
agents are well known in the art, and are disclosed in U.S. Pat. No.
4,676,980, along with a number of cross-linking techniques.

[0258]Techniques for generating bispecific antibodies from antibody
fragments have also been described in the literature. For example,
bispecific antibodies can be prepared using chemical linkage. Brennan et
al., Science, 229: 81 (1985) describe a procedure wherein intact
antibodies are proteolytically cleaved to generate F(ab')2
fragments. These fragments are reduced in the presence of the dithiol
complexing agent sodium arsenite to stabilize vicinal dithiols and
prevent intermolecular disulfide formation. The Fab' fragments generated
are then converted to thionitrobenzoate (TNB) derivatives. One of the
Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction
with mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The bispecific
antibodies produced can be used as agents for the selective
immobilization of enzymes.

[0259]Recent progress has facilitated the direct recovery of Fab'-SH
fragments from E. coli, which can be chemically coupled to form
bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992)
describe the production of a fully humanized bispecific antibody
F(ab')2 molecule. Each Fab' fragment was separately secreted from E.
coli and subjected to directed chemical coupling in vitro to form the
bispecific antibody. The bispecific antibody thus formed was able to bind
to cells overexpressing the HER2 receptor and normal human T cells, as
well as trigger the lytic activity of human cytotoxic lymphocytes against
human breast tumor targets.

[0260]Various techniques for making and isolating bispecific antibody
fragments directly from recombinant cell culture have also been
described. For example, bispecific antibodies have been produced using
leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).
The leucine zipper peptides from the Fos and Jun proteins were linked to
the Fab' portions of two different antibodies by gene fusion. The
antibody homodimers were reduced at the hinge region to form monomers and
then re-oxidized to form the antibody heterodimers. This method can also
be utilized for the production of antibody homodimers. The "diabody"
technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA,
90:6444-6448 (1993) has provided an alternative mechanism for making
bispecific antibody fragments. The fragments comprise a heavy-chain
variable domain (VH) connected to a light-chain variable domain (VL) by a
linker which is too short to allow pairing between the two domains on the
same chain. Accordingly, the VH and VL domains of one fragment are forced
to pair with the complementary VL and VH domains of another fragment,
thereby forming two antigen-binding sites. Another strategy for making
bispecific antibody fragments by the use of single-chain Fv (sFv) dimers
has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0261]Antibodies with more than two valencies are contemplated. For
example, trispecific antibodies can be prepared. Tuft et al. J. Immunol.
147: 60 (1991).

Multivalent Antibodies

[0262]A multivalent antibody may be internalized (and/or catabolized)
faster than a bivalent antibody by a cell expressing an antigen to which
the antibodies bind. The antibodies of the present invention can be
multivalent antibodies (which are other than of the IgM class) with three
or more antigen binding sites (e.g. tetravalent antibodies), which can be
readily produced by recombinant expression of nucleic acid encoding the
polypeptide chains of the antibody. The multivalent antibody can comprise
a dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc region or
a hinge region. In this scenario, the antibody will comprise an Fc region
and three or more antigen binding sites amino-terminal to the Fe region.
The preferred multivalent antibody herein comprises (or consists of)
three to about eight, but preferably four, antigen binding sites. The
multivalent antibody comprises at least one polypeptide chain (and
preferably two polypeptide chains), wherein the polypeptide chain(s)
comprise two or more variable domains. For instance, the polypeptide
chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first
variable domain, VD2 is a second variable domain, Fc is one polypeptide
chain of an Fc region, X1 and X2 represent an amino acid or polypeptide,
and n is 0 or 1. For instance, the polypeptide chain(s) may comprise:
VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region
chain. The multivalent antibody herein preferably further comprises at
least two (and preferably four) light chain variable domain polypeptides.
The multivalent antibody herein may, for instance, comprise from about
two to about eight light chain variable domain polypeptides. The light
chain variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL domain.

Antibody Variants

[0263]In some embodiments, amino acid sequence modification(s) of the
antibodies described herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of the antibody
are prepared by introducing appropriate nucleotide changes into the
antibody nucleic acid, or by peptide synthesis. Such modifications
include, for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
antibody. Any combination of deletion, insertion, and substitution is
made to arrive at the final construct, provided that the final construct
possesses the desired characteristics. The amino acid alterations may be
introduced in the subject antibody amino acid sequence at the time that
sequence is made.

[0264]A useful method for identification of certain residues or regions of
the antibody that are preferred locations for mutagenesis is called
"alanine scanning mutagenesis" as described by Cunningham and Wells
(1989) Science, 244:1081-1085. Here, a residue or group of target
residues are identified (e.g., charged residues such as arg, asp, his,
lys, and glu) and replaced by a neutral or negatively charged amino acid
(most preferably alanine or polyalanine) to affect the interaction of the
amino acids with antigen. Those amino acid locations demonstrating
functional sensitivity to the substitutions then are refined by
introducing further or other variants at, or for, the sites of
substitution. Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need not be
predetermined. For example, to analyze the performance of a mutation at a
given site, ala scanning or random mutagenesis is conducted at the target
codon or region and the expressed immunoglobulins are screened for the
desired activity.

[0265]Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an N-terminal
methionyl residue or the antibody fused to a cytotoxic polypeptide. Other
insertional variants of the antibody molecule include the fusion to the
N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a
polypeptide which increases the serum half-life of the antibody.

[0266]Glycosylation of polypeptides is typically either N-linked or
O-linked. N-linked refers to the attachment of the carbohydrate moiety to
the side chain of an asparagine residue. The tripeptide sequences
asparagine-X-serine and asparagine-X-threonine, where X is any amino acid
except proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the presence
of either of these tripeptide sequences in a polypeptide creates a
potential glycosylation site. O-linked glycosylation refers to the
attachment of one of the sugars N-aceylgalactosamine, galactose, or
xylose to a hydroxyamino acid, most commonly serine or threonine,
although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0267]Addition of glycosylation sites to the antibody is conveniently
accomplished by altering the amino acid sequence such that it contains
one or more of the above-described tripeptide sequences (for N-linked
glycosylation sites). The alteration may also be made by the addition of,
or substitution by, one or more serine or threonine residues to the
sequence of the original antibody (for O-linked glycosylation sites).

[0268]Where the antibody comprises an Fc region, the carbohydrate attached
thereto may be altered. For example, antibodies with a mature
carbohydrate structure that lacks fucose attached to an Fc region of the
antibody are described in US Pat Appl No US 2003/0157108 (Presta, L.).
See also US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Antibodies with a
bisecting N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an
Fc region of the antibody are referenced in WO 2003/011878, Jean-Mairet
et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at least
one galactose residue in the oligosaccharide attached to an Fc region of
the antibody are reported in WO 1997/30087, Patel et al. See, also, WO
1998/58964 (Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies
with altered carbohydrate attached to the Fc region thereof. See also US
2005/0123546 (Umana et al.) on antigen-binding molecules with modified
glycosylation.

[0270]Another type of variant is an amino acid substitution variant. These
variants have at least one amino acid residue in the antibody molecule
replaced by a different residue. The sites of greatest interest for
substitutional mutagenesis include the hypervariable regions, but FR
alterations are also contemplated. Conservative substitutions are shown
in Table 1 under the heading of "preferred substitutions". If such
substitutions result in a change in biological activity, then more
substantial changes, denominated "exemplary substitutions" in Table 1, or
as further described below in reference to amino acid classes, may be
introduced and the products screened.

[0271]Substantial modifications in the biological properties of the
antibody are accomplished by selecting substitutions that differ
significantly in their effect on maintaining (a) the structure of the
polypeptide backbone in the area of the substitution, for example, as a
sheet or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain. Naturally
occurring residues are divided into groups based on common side-chain
properties: [0272](1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0273](2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; [0274](3) acidic:
asp, glu; [0275](4) basic: his, lys, arg; [0276](5) residues that
influence chain orientation: gly, pro; and [0277](6) aromatic: trp, tyr,
phe.

[0278]Non-conservative substitutions will entail exchanging a member of
one of these classes for another class.

[0279]One type of substitutional variant involves substituting one or more
hypervariable region residues of a parent antibody (e.g. a humanized or
human antibody). Generally, the resulting variant(s) selected for further
development will have improved biological properties relative to the
parent antibody from which they are generated. A convenient way for
generating such substitutional variants involves affinity maturation
using phage display. Briefly, several hypervariable region sites (e.g.
6-7 sites) are mutated to generate all possible amino acid substitutions
at each site. The antibodies thus generated are displayed from
filamentous phage particles as fusions to the gene III product of M13
packaged within each particle. The phage-displayed variants are then
screened for their biological activity (e.g. binding affinity) as herein
disclosed. In order to identify candidate hypervariable region sites for
modification, alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to analyze
a crystal structure of the antigen-antibody complex to identify contact
points between the antibody and antigen. Such contact residues and
neighboring residues are candidates for substitution according to the
techniques elaborated herein. Once such variants are generated, the panel
of variants is subjected to screening as described herein and antibodies
with superior properties in one or more relevant assays may be selected
for further development.

[0280]Nucleic acid molecules encoding amino acid sequence variants of the
antibody are prepared by a variety of methods known in the art. These
methods include, but are not limited to, isolation from a natural source
(in the case of naturally occurring amino acid sequence variants) or
preparation by oligonucleotide-mediated (or site-directed) mutagenesis,
PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant
or a non-variant version of the antibody.

[0281]It may be desirable to introduce one or more amino acid
modifications in an Fc region of the immunoglobulin polypeptides of the
invention, thereby generating a Fc region variant. The Fc region variant
may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3
or IgG4 Fc region) comprising an amino acid modification (e.g. a
substitution) at one or more amino acid positions including that of a
hinge cysteine. In accordance with this description and the teachings of
the art, it is contemplated that in some embodiments, an antibody used in
methods of the invention may comprise one or more alterations as compared
to the wild type counterpart antibody, e.g. in the Fc region. These
antibodies would nonetheless retain substantially the same
characteristics required for therapeutic utility as compared to their
wild type counterpart. For example, it is thought that certain
alterations can be made in the Fc region that would result in altered
(i.e., either improved or diminished) Clq binding and/or Complement
Dependent Cytotoxicity (CDC), e.g., as described in WO99/51642. See also
Duncan & Winter Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S.
Pat. No. 5,624,821; and WO94/29351 concerning other examples of Fc region
variants. WO00/42072 (Presta) and WO 2004/056312 (Lowman) describe
antibody variants with improved or diminished binding to FcRs. The
content of these patent publications are specifically incorporated herein
by reference. See, also, Shields et al. J. Biol. Chem. 9(2): 6591-6604
(2001). Antibodies with increased half lives and improved binding to the
neonatal Fc receptor (FcRn), which is responsible for the transfer of
maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and
Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934A1
(Hinton et al.). These antibodies comprise an Fc reg on with one or more
substitutions therein which improve binding of the Fc region to FcRn.
Polypeptide variants with altered Fc region amino acid sequences and
increased or decreased Clq binding capability are described in U.S. Pat.
No. 6,194,551B1, WO99/51642. The contents of those patent publications
are specifically incorporated herein by reference. See, also, Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).

Antibody Derivatives

[0282]The antibodies of the present invention can be further modified to
contain additional nonproteinaceous moieties that are known in the art
and readily available. Preferably, the moieties suitable for
derivatization of the antibody are water soluble polymers. Non-limiting
examples of water soluble polymers include, but are not limited to,
polyethylene glycol (PEG), copolymers of ethylene glycol/propylene
glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic
anhydride copolymer, polyaminoacids (either homopolymers or random
copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol,
propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide
co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl
alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may
have advantages in manufacturing due to its stability in water. The
polymer may be of any molecular weight, and may be branched or
unbranched. The number of polymers attached to the antibody may vary, and
if more than one polymers are attached, they can be the same or different
molecules. In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including, but
not limited to, the particular properties or functions of the antibody to
be improved, whether the antibody derivative will be used in a therapy
under defined conditions, etc.

Screening for Antibodies with Desired Properties

[0283]The antibodies of the present invention can be characterized for
their physical/chemical properties and biological functions by various
assays known in the art. In some embodiments, antibodies are
characterized for any one or more of binding to DLL4; and/or reduction or
blocking of Notch receptor activation; and/or reduction or blocking of
Notch receptor downstream molecular signaling; and/or disruption or
blocking of Notch receptor binding to DLL4; and/or promotion of
endothelial cell proliferation; and/or inhibition of endothelial cell
differentiation; and/or inhibition of arterial differentiation; and/or
inhibition of tumor vascular perfusion; and/or treatment and/or
prevention of a tumor, cell proliferative disorder or a cancer; and/or
treatment or prevention of a disorder associated with DLL4 expression
and/or activity; and/or treatment or prevention of a disorder associated
with Notch receptor expression and/or activity.

[0285]In certain embodiments of the invention, the antibodies produced
herein are analyzed for their biological activity. In some embodiments,
the antibodies of the present invention are tested for their antigen
binding activity. The antigen binding assays that are known in the art
and can be used herein include without limitation any direct or
competitive binding assays using techniques such as western blots,
radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich"
immunoassays, immunoprecipitation assays, fluorescent immunoassays, and
protein A immunoassays. Illustrative antigen binding assay are provided
below in the Examples section.

[0286]In still another embodiment, the invention provides anti-DLL4
monoclonal antibodies that compete with a 26.6, 26.14, 26.20, 26.34,
and/or 26.82 antibody for binding to DLL4. Such competitor antibodies
include antibodies that recognize an DLL4 epitope that is the same as or
overlaps with the DLL4 epitope recognized by antibody 26.6, 26.14, 26.20,
26.34, and/or 26.82. Such competitor antibodies can be obtained by
screening anti-DLL4 hybridoma supernatants for binding to immobilized
DLL4 in competition with labeled 26.6, 26.14, 26.20, 26.34, and/or 26.82
antibody. A hybridoma supernatant containing competitor antibody will
reduce the amount of bound, labeled antibody detected in the subject
competition binding mixture as compared to the amount of bound, labeled
antibody detected in a control binding mixture containing irrelevant (or
no) antibody. Any of the competition binding assays described herein are
suitable for use in the foregoing procedure.

[0287]In another aspect, the invention provides an anti-DLL4 monoclonal
antibody that comprises one or more (such as 2, 3, 4, 5, and/or 6) HVRs
of an 26.6, 26.14, 26.20, 26.34, and/or 26.82 antibody. An anti-DLL4
monoclonal antibody that comprises one or more HVR(s) of an 26.6, 26.14,
26.20, 26.34, and/or 26.82 antibody can be constructed by grafting one or
more HVR(s) of an 26.6, 26.14, 26.20, 26.34, and/or 26.82 antibody onto a
template antibody sequence, e.g. a human antibody sequence which is
closest to the corresponding murine sequence of the parental antibody, or
a consensus sequence of all human antibodies in the particular subgroup
of the parental antibody light or heavy chain, and expressing the
resulting chimeric light and/or heavy chain variable region sequence(s),
with or without accompanying constant region sequence(s), in recombinant
host cells as described herein.

[0288]Anti-DLL4 antibodies of the invention possessing the unique
properties described herein can be obtained by screening anti-DLL4
hybridoma clones for the desired properties by any convenient method. For
example, if an anti-DLL4 monoclonal antibody that blocks or does not
block the binding of Notch receptors to DLL4 is desired, the candidate
antibody can be tested in a binding competition assay, such as a
competitive binding ELISA, wherein plate wells are coated with DLL4, and
a solution of antibody in an excess of the Notch receptor of interest is
layered onto the coated plates, and bound antibody is detected
enzymatically, e.g. contacting the bound antibody with HRP-conjugated
anti-Ig antibody or biotinylated anti-Ig antibody and developing the HRP
color reaction., e.g. by developing plates with streptavidin-HRP and/or
hydrogen peroxide and detecting the HRP color reaction by
spectrophotometry at 490 nm with an ELISA plate reader.

[0289]In one embodiment, the present invention contemplates an altered
antibody that possesses some but not all effector functions, which make
it a desired candidate for many applications in which the half life of
the antibody in vivo is important yet certain effector functions (such as
complement and ADCC) are unnecessary or deleterious. In certain
embodiments, the Fc activities of the produced immunoglobulin are
measured to ensure that only the desired properties are maintained. In
vitro and/or in vivo cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
antibody lacks FcγR binding (hence likely lacking ADCC activity),
but retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express FcγRIII only, whereas monocytes express

[0290]FcγRI, FcγRII and FcγRIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and
Kinet, Annu. Rev. Immunol 9:457-92 (1991). An example of an in vitro
assay to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for such
assays include peripheral blood mononuclear cells (PBMC) and Natural
Killer (NK) cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in a animal model
such as that disclosed in Clynes et al. Proc. Natl. Acad. Sci. A
95:652-656 (1998). Clq binding assays may also be carried out to confirm
that the antibody is unable to bind Clq and hence lacks CDC activity. To
assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed. FcRn binding and in vivo clearance/half life determinations
can also be performed using methods known in the art, e.g. those
described in the Examples section.

Vectors, Host Cells and Recombinant Methods

[0291]For recombinant production of an antibody of the invention, the
nucleic acid encoding it is isolated and inserted into a replicable
vector for further cloning (amplification of the DNA) or for expression.
DNA encoding the antibody is readily isolated and sequenced using
conventional procedures (e.g., by using oligonucleotide probes that are
capable of binding specifically to genes encoding the heavy and light
chains of the antibody). Many vectors are available. The choice of vector
depends in part on the host cell to be used. Generally, preferred host
cells are of either prokaryotic or eukaryotic (generally mammalian)
origin. It will be appreciated that constant regions of any isotype can
be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant
regions, and that such constant regions can be obtained from any human or
animal species.

[0292]a. Generating Antibodies Using Prokaryotic Host Cells:

[0293]i. Vector Construction

[0294]Polynucleotide sequences encoding polypeptide components of the
antibody of the invention can be obtained using standard recombinant
techniques. Desired polynucleotide sequences may be isolated and
sequenced from antibody producing cells such as hybridoma cells.
Alternatively, polynucleotides can be synthesized using nucleotide
synthesizer or PCR techniques. Once obtained, sequences encoding the
polypeptides are inserted into a recombinant vector capable of
replicating and expressing heterologous polynucleotides in prokaryotic
hosts. Many vectors that are available and known in the art can be used
for the purpose of the present invention. Selection of an appropriate
vector will depend mainly on the size of the nucleic acids to be inserted
into the vector and the particular host cell to be transformed with the
vector. Each vector contains various components, depending on its
function (amplification or expression of heterologous polynucleotide, or
both) and its compatibility with the particular host cell in which it
resides. The vector components generally include, but are not limited to:
an origin of replication, a selection marker gene, a promoter, a ribosome
binding site (RBS), a signal sequence, the heterologous nucleic acid
insert and a transcription termination sequence.

[0295]In general, plasmid vectors containing replicon and control
sequences which are derived from species compatible with the host cell
are used in connection with these hosts. The vector ordinarily carries a
replication site, as well as marking sequences which are capable of
providing phenotypic selection in transformed cells. For example, E. coli
is typically transformed using pBR322, a plasmid derived from an E. coli
species. pBR322 contains genes encoding ampicillin (Amp) and tetracycline
(Tet) resistance and thus provides easy means for identifying transformed
cells. pBR322, its derivatives, or other microbial plasmids or
bacteriophage may also contain, or be modified to contain, promoters
which can be used by the microbial organism for expression of endogenous
proteins. Examples of pBR322 derivatives used for expression of
particular antibodies are described in detail in Carter et al., U.S. Pat.
No. 5,648,237.

[0296]In addition, phage vectors containing replicon and control sequences
that are compatible with the host microorganism can be used as
transforming vectors in connection with these hosts. For example,
bacteriophage such as λGEM®-11 may be utilized in making a
recombinant vector which can be used to transform susceptible host cells
such as E. coli LE392.

[0297]The expression vector of the invention may comprise two or more
promoter-cistron pairs, encoding each of the polypeptide components. A
promoter is an untranslated regulatory sequence located upstream (5') to
a cistron that modulates its expression. Prokaryotic promoters typically
fall into two classes, inducible and constitutive. Inducible promoter is
a promoter that initiates increased levels of transcription of the
cistron under its control in response to changes in the culture
condition, e.g. the presence or absence of a nutrient or a change in
temperature.

[0298]A large number of promoters recognized by a variety of potential
host cells are well known. The selected promoter can be operably linked
to cistron DNA encoding the light or heavy chain by removing the promoter
from the source DNA via restriction enzyme digestion and inserting the
isolated promoter sequence into the vector of the invention. Both the
native promoter sequence and many heterologous promoters may be used to
direct amplification and/or expression of the target genes. In some
embodiments, heterologous promoters are utilized, as they generally
permit greater transcription and higher yields of expressed target gene
as compared to the native target polypeptide promoter.

[0299]Promoters suitable for use with prokaryotic hosts include the PhoA
promoter, the β-galactamase and lactose promoter systems, a
tryptophan (trp) promoter system and hybrid promoters such as the tac or
the trc promoter. However, other promoters that are functional in
bacteria (such as other known bacterial or phage promoters) are suitable
as well. Their nucleotide sequences have been published, thereby enabling
a skilled worker operably to ligate them to cistrons encoding the target
light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using
linkers or adaptors to supply any required restriction sites.

[0300]In one aspect of the invention, each cistron within the recombinant
vector comprises a secretion signal sequence component that directs
translocation of the expressed polypeptides across a membrane. In
general, the signal sequence may be a component of the vector, or it may
be a part of the target polypeptide DNA that is inserted into the vector.
The signal sequence selected for the purpose of this invention should be
one that is recognized and processed (i.e. cleaved by a signal peptidase)
by the host cell. For prokaryotic host cells that do not recognize and
process the signal sequences native to the heterologous polypeptides, the
signal sequence is substituted by a prokaryotic signal sequence selected,
for example, from the group consisting of the alkaline phosphatase,
penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB,
PhoE, PeIB, OmpA and MBP. In one embodiment of the invention, the signal
sequences used in both cistrons of the expression system are STII signal
sequences or variants thereof.

[0301]In another aspect, the production of the immunoglobulins according
to the invention can occur in the cytoplasm of the host cell, and
therefore does not require the presence of secretion signal sequences
within each cistron. In that regard, immunoglobulin light and heavy
chains are expressed, folded and assembled to form functional
immunoglobulins within the cytoplasm. Certain host strains (e.g., the E.
coli trxB-strains) provide cytoplasm conditions that are favorable for
disulfide bond formation, thereby permitting proper folding and assembly
of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0302]Prokaryotic host cells suitable for expressing antibodies of the
invention include Archaebacteria and Eubacteria, such as Gram-negative or
Gram-positive organisms. Examples of useful bacteria include Escherichia
(e.g., E. coli), Bacilli (e.g., B. subtilis), Enterobacteria, Pseudomonas
species (e.g., P. aeruginosa), Salmonella typhimurium, Serratia
marcescans, Klebsiella, Proteus, Shigella, Rhizobia, Vitreoscilla, or
Paracoccus. In one embodiment, gram-negative cells are used. In one
embodiment, E. coli cells are used as hosts for the invention. Examples
of E. coli strains include strain W3110 (Bachmann, Cellular and Molecular
Biology, vol. 2 (Washington, D.C.: American Society for Microbiology,
1987), pp. 1190-1219; ATCC® Deposit No. 27,325) and derivatives
thereof, including strain 33D3 having genotype W3110 ΔfhuA
(ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41 kanR
(U.S. Pat. No. 5,639,635). Other strains and derivatives thereof, such as
E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776 (ATCC 31,537)
and E. coli RV308(ATCC 31,608) are also suitable. These examples are
illustrative rather than limiting. Methods for constructing derivatives
of any of the above-mentioned bacteria having defined genotypes are known
in the art and described in, for example, Bass et al., Proteins,
8:309-314 (1990). It is generally necessary to select the appropriate
bacteria taking into consideration replicability of the replicon in the
cells of a bacterium. For example, E. coli, Serratia, or Salmonella
species can be suitably used as the host when well known plasmids such as
pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon.
Typically the host cell should secrete minimal amounts of proteolytic
enzymes, and additional protease inhibitors may desirably be incorporated
in the cell culture.

[0303]ii. Antibody Production

[0304]Host cells are transformed with the above-described expression
vectors and cultured in conventional nutrient media modified as
appropriate for inducing promoters, selecting transformants, or
amplifying the genes encoding the desired sequences.

[0305]Transformation means introducing DNA into the prokaryotic host so
that the DNA is replicable, either as an extrachromosomal element or by
chromosomal integrant. Depending on the host cell used, transformation is
done using standard techniques appropriate to such cells. The calcium
treatment employing calcium chloride is generally used for bacterial
cells that contain substantial cell-wall barriers. Another method for
transformation employs polyethylene glycol/DMSO. Yet another technique
used is electroporation.

[0306]Prokaryotic cells used to produce the polypeptides of the invention
are grown in media known in the art and suitable for culture of the
selected host cells. Examples of suitable media include luria broth (LB)
plus necessary nutrient supplements. In some embodiments, the media also
contains a selection agent, chosen based on the construction of the
expression vector, to selectively permit growth of prokaryotic cells
containing the expression vector. For example, ampicillin is added to
media for growth of cells expressing ampicillin resistant gene.

[0307]Any necessary supplements besides carbon, nitrogen, and inorganic
phosphate sources may also be included at appropriate concentrations
introduced alone or as a mixture with another supplement or medium such
as a complex nitrogen source. Optionally the culture medium may contain
one or more reducing agents selected from the group consisting of
glutathione, cysteine, cystamine, thioglycollate, dithioerythritol and
dithiothreitol.

[0308]The prokaryotic host cells are cultured at suitable temperatures.
For E. coli growth, for example, the preferred temperature ranges from
about 20° C. to about 39° C., more preferably from about
25° C. to about 37° C., even more preferably at about
30° C. The pH of the medium may be any pH ranging from about 5 to
about 9, depending mainly on the host organism. For E. coli, the pH is
preferably from about 6.8 to about 7.4, and more preferably about 7.0.

[0309]If an inducible promoter is used in the expression vector of the
invention, protein expression is induced under conditions suitable for
the activation of the promoter. In one aspect of the invention, PhoA
promoters are used for controlling transcription of the polypeptides.
Accordingly, the transformed host cells are cultured in a
phosphate-limiting medium for induction. Preferably, the
phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons et
al., J. Immunol. Methods (2002), 263:133-147). A variety of other
inducers may be used, according to the vector construct employed, as is
known in the art.

[0310]In one embodiment, the expressed polypeptides of the present
invention are secreted into and recovered from the periplasm of the host
cells. Protein recovery typically involves disrupting the microorganism,
generally by such means as osmotic shock, sonication or lysis. Once cells
are disrupted, cell debris or whole cells may be removed by
centrifugation or filtration. The proteins may be further purified, for
example, by affinity resin chromatography. Alternatively, proteins can be
transported into the culture media and isolated therein. Cells may be
removed from the culture and the culture supernatant being filtered and
concentrated for further purification of the proteins produced. The
expressed polypeptides can be further isolated and identified using
commonly known methods such as polyacrylamide gel electrophoresis (PAGE)
and Western blot assay.

[0311]In one aspect of the invention, antibody production is conducted in
large quantity by a fermentation process. Various large-scale fed-batch
fermentation procedures are available for production of recombinant
proteins. Large-scale fermentations have at least 1000 liters of
capacity, preferably about 1,000 to 100,000 liters of capacity. These
fermentors use agitator impellers to distribute oxygen and nutrients,
especially glucose (the preferred carbon/energy source). Small scale
fermentation refers generally to fermentation in a fermentor that is no
more than approximately 100 liters in volumetric capacity, and can range
from about 1 liter to about 100 liters.

[0312]In a fermentation process, induction of protein expression is
typically initiated after the cells have been grown under suitable
conditions to a desired density, e.g., an OD550 of about 180-220, at
which stage the cells are in the early stationary phase. A variety of
inducers may be used, according to the vector construct employed, as is
known in the art and described above. Cells may be grown for shorter
periods prior to induction. Cells are usually induced for about 12-50
hours, although longer or shorter induction time may be used.

[0313]To improve the production yield and quality of the polypeptides of
the invention, various fermentation conditions can be modified. For
example, to improve the proper assembly and folding of the secreted
antibody polypeptides, additional vectors overexpressing chaperone
proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or
FkpA (a peptidylprolyl cis,trans-isomerase with chaperone activity) can
be used to co-transform the host prokaryotic cells. The chaperone
proteins have been demonstrated to facilitate the proper folding and
solubility of heterologous proteins produced in bacterial host cells.
Chen et al. (1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat.
No. 6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann and
Pluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun
(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol.
39:199-210.

[0315]In one embodiment, E. coli strains deficient for proteolytic enzymes
and transformed with plasmids overexpressing one or more chaperone
proteins are used as host cells in the expression system of the
invention.

[0316]iii. Antibody Purification

[0317]Standard protein purification methods known in the art can be
employed. The following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange columns,
ethanol precipitation, reverse phase HPLC, chromatography on silica or on
a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,
ammonium sulfate precipitation, and gel filtration using, for example,
Sephadex G-75.

[0318]In one aspect, Protein A immobilized on a solid phase is used for
immunoaffinity purification of the full length antibody products of the
invention. Protein A is a 41 kD cell wall protein from Staphylococcus
aureas which binds with a high affinity to the Fc region of antibodies.
Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase to which
Protein A is immobilized is preferably a column comprising a glass or
silica surface, more preferably a controlled pore glass column or a
silicic acid column. In some applications, the column has been coated
with a reagent, such as glycerol, in an attempt to prevent nonspecific
adherence of contaminants.

[0319]As the first step of purification, the preparation derived from the
cell culture as described above is applied onto the Protein A immobilized
solid phase to allow specific binding of the antibody of interest to
Protein A. The solid phase is then washed to remove contaminants
non-specifically bound to the solid phase. Finally the antibody of
interest is recovered from the solid phase by elution.

[0320]b. Generating Antibodies Using Eukaryotic Host Cells:

[0321]The vector components generally include, but are not limited to, one
or more of the following: a signal sequence, an origin of replication,
one or more marker genes, an enhancer element, a promoter, and a
transcription termination sequence.

[0322](i) Signal Sequence Component

[0323]A vector for use in a eukaryotic host cell may also contain a signal
sequence or other polypeptide having a specific cleavage site at the
N-terminus of the mature protein or polypeptide of interest. The
heterologous signal sequence selected preferably is one that is
recognized and processed (i.e., cleaved by a signal peptidase) by the
host cell. In mammalian cell expression, mammalian signal sequences as
well as viral secretory leaders, for example, the herpes simplex gD
signal, are available.

[0324]The DNA for such precursor region is ligated in reading frame to DNA
encoding the antibody.

[0325](ii) Origin of Replication

[0326]Generally, an origin of replication component is not needed for
mammalian expression vectors. For example, the SV40 origin may typically
be used only because it contains the early promoter.

[0327](iii) Selection Gene Component

[0328]Expression and cloning vectors may contain a selection gene, also
termed a selectable marker. Typical selection genes encode proteins that
(a) confer resistance to antibiotics or other toxins, e.g., ampicillin,
neomycin, methotrexate, or tetracycline, (b) complement auxotrophic
deficiencies, where relevant, or (c) supply critical nutrients not
available from complex media.

[0329]One example of a selection scheme utilizes a drug to arrest growth
of a host cell. Those cells that are successfully transformed with a
heterologous gene produce a protein conferring drug resistance and thus
survive the selection regimen. Examples of such dominant selection use
the drugs neomycin, mycophenolic acid and hygromycin.

[0330]Another example of suitable selectable markers for mammalian cells
are those that enable the identification of cells competent to take up
the antibody nucleic acid, such as DHFR, thymidine kinase,
metallothionein-I and -II, preferably primate metallothionein genes,
adenosine deaminase, ornithine decarboxylase, etc.

[0331]For example, cells transformed with the DHFR selection gene are
first identified by culturing all of the transformants in a culture
medium that contains methotrexate (Mtx), a competitive antagonist of
DHFR. An appropriate host cell when wild-type DHFR is employed is the
Chinese hamster ovary (CHO) cell line deficient in DHFR activity (e.g.,
ATCC CRL-9096).

[0332]Alternatively, host cells (particularly wild-type hosts that contain
endogenous DHFR) transformed or co-transformed with DNA sequences
encoding an antibody, wild-type DHFR protein, and another selectable
marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected
by cell growth in medium containing a selection agent for the selectable
marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin,
or G418. See U.S. Pat. No. 4,965,199.

[0333](iv) Promoter Component

[0334]Expression and cloning vectors usually contain a promoter that is
recognized by the host organism and is operably linked to the antibody
polypeptide nucleic acid. Promoter sequences are known for eukaryotes.
Virtually alleukaryotic genes have an AT-rich region located
approximately 25 to 30 bases upstream from the site where transcription
is initiated. Another sequence found 70 to 80 bases upstream from the
start of transcription of many genes is a CNCAAT region where N may be
any nucleotide (SEQ ID NO: 60). At the 3' end of most eukaryotic genes is
an AATAAA sequence that may be the signal for addition of the poly A tail
to the 3' end of the coding sequence (SEQ ID NO: 61). All of these
sequences are suitably inserted into eukaryotic expression vectors.

[0335]Antibody polypeptide transcription from vectors in mammalian host
cells is controlled, for example, by promoters obtained from the genomes
of viruses such as polyoma virus, fowlpox virus, adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus,
cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40
(SV40), from heterologous mammalian promoters, e.g., the actin promoter
or an immunoglobulin promoter, from heat-shock promoters, provided such
promoters are compatible with the host cell systems.

[0336]The early and late promoters of the SV40 virus are conveniently
obtained as an SV40 restriction fragment that also contains the SV40
viral origin of replication. The immediate early promoter of the human
cytomegalovirus is conveniently obtained as a HindIII E restriction
fragment. A system for expressing DNA in mammalian hosts using the bovine
papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A
modification of this system is described in U.S. Pat. No. 4,601,978.
Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as
the promoter.

[0337](v) Enhancer Element Component

[0338]Transcription of DNA encoding the antibody polypeptide of this
invention by higher eukaryotes is often increased by inserting an
enhancer sequence into the vector. Many enhancer sequences are now known
from mammalian genes (globin, elastase, albumin, α-fetoprotein, and
insulin). Typically, however, one will use an enhancer from a eukaryotic
cell virus. Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982)
on enhancing elements for activation of eukaryotic promoters. The
enhancer may be spliced into the vector at a position 5' or 3' to the
antibody polypeptide-encoding sequence, but is preferably located at a
site 5' from the promoter.

[0339](vi) Transcription Termination Component

[0340]Expression vectors used in eukaryotic host cells will typically also
contain sequences necessary for the termination of transcription and for
stabilizing the mRNA. Such sequences are commonly available from the 5'
and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or
cDNAs. These regions contain nucleotide segments transcribed as
polyadenylated fragments in the untranslated portion of the mRNA encoding
an antibody. One useful transcription termination component is the bovine
growth hormone polyadenylation region. See WO94/11026 and the expression
vector disclosed therein.

[0343]Host cells are transformed with the above-described expression or
cloning vectors for antibody production and cultured in conventional
nutrient media modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired sequences.

[0344](viii) Culturing the Host Cells

[0345]The host cells used to produce an antibody of this invention may be
cultured in a variety of media. Commercially available media such as
Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640
(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are
suitable for culturing the host cells. In addition, any of the media
described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.
Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;
4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.
30,985 may be used as culture media for the host cells. Any of these
media may be supplemented as necessary with hormones and/or other growth
factors (such as insulin, transferrin, or epidermal growth factor), salts
(such as sodium chloride, calcium, magnesium, and phosphate), buffers
(such as HEPES), nucleotides (such as adenosine and thymidine),
antibiotics (such as GENTAMYCINT® drug), trace elements (defined as
inorganic compounds usually present at final concentrations in the
micromolar range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate concentrations
that would be known to those skilled in the art. The culture conditions,
such as temperature, pH, and the like, are those previously used with the
host cell selected for expression, and will be apparent to the ordinarily
skilled artisan.

[0346](ix) Purification of Antibody

[0347]When using recombinant techniques, the antibody can be produced
intracellularly, or directly secreted into the medium. If the antibody is
produced intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Where the antibody is secreted into
the medium, supernatants from such expression systems are generally first
concentrated using a commercially available protein concentration filter,
for example, an Amicon or Millipore Pellicon® ultrafiltration unit. A
protease inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to prevent
the growth of adventitious contaminants.

[0348]The antibody composition prepared from the cells can be purified
using, for example, hydroxylapatite chromatography, gel electrophoresis,
dialysis, and affinity chromatography, with affinity chromatography being
the preferred purification technique. The suitability of protein A as an
affinity ligand depends on the species and isotype of any immunoglobulin
Fc domain that is present in the antibody. Protein A can be used to
purify antibodies that are based on human γ1, γ2, or γ4
heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein
G is recommended for all mouse isotypes and for human γ3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand
is attached is most often agarose, but other matrices are available.
Mechanically stable matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the antibody
comprises a CH3 domain, the Bakerbond ABX® resin (J. T. Baker,
Phillipsburg, N.J.) is useful for purification. Other techniques for
protein purification such as fractionation on an ion-exchange column,
ethanol precipitation, Reverse Phase HPLC, chromatography on silica,
chromatography on heparin SEPHAROSE® chromatography on an anion or
cation exchange resin (such as a polyaspartic acid column),
chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also
available depending on the antibody to be recovered.

[0349]Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be subjected to
low pH hydrophobic interaction chromatography using an elution buffer at
a pH between about 2.5-4.5, preferably performed at low salt
concentrations (e.g., from about 0-0.25 M salt).

Immunoconjugates

[0350]The invention also provides immunoconjugates (interchangeably termed
"antibody-drug conjugates" or "ADC"), comprising any of the anti-DLL4
antibodies described herein conjugated to a cytotoxic agent such as a
chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g.,
an enzymatically active toxin of bacterial, fungal, plant, or animal
origin, or fragments thereof), or a radioactive isotope (i.e., a
radioconjugate).

[0353]Chemotherapeutic agents useful in the generation of immunoconjugates
are described herein (eg., above). Enzymatically active toxins and
fragments thereof that can be used include diphtheria A chain, nonbinding
active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas
aeruginosa), ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca
americana proteins (PAPI, PAPII, and PAP-S), momordica charantia
inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
See, e.g., WO 93/21232 published Oct. 28, 1993. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include 212Bi, 131I, 131In, 90Y,
and 186Re. Conjugates of the antibody and cytotoxic agent are made
using a variety of bifunctional protein-coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane
(IT), bifunctional derivatives of imidoesters (such as dimethyl
adipimidate HCl), active esters (such as disuccinimidyl suberate),
aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis
(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene
2,6-diisocyanate), and bis-active fluorine compounds (such as
1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be
prepared as described in Vitetta et al., Science, 238: 1098 (1987).
Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for
conjugation of radionucleotide to the antibody. See WO94/11026.

[0354]Conjugates of an antibody and one or more small molecule toxins,
such as a calicheamicin, maytansinoids, dolastatins, aurostatins, a
trichothecene, and CC1065, and the derivatives of these toxins that have
toxin activity, are also contemplated herein.

[0355]i. Maytansine and Maytansinoids

[0356]In some embodiments, the immunoconjugate comprises an antibody (full
length or fragments) of the invention conjugated to one or more
maytansinoid molecules.

[0358]Maytansinoid drug moieties are attractive drug moieties in antibody
drug conjugates because they are: (i) relatively accessible to prepare by
fermentation or chemical modification, derivatization of fermentation
products, (ii) amenable to derivatization with functional groups suitable
for conjugation through the non-disulfide linkers to antibodies, (iii)
stable in plasma, and (iv) effective against a variety of tumor cell
lines.

[0359]Immunoconjugates containing maytansinoids, methods of making same,
and their therapeutic use are disclosed, for example, in U.S. Pat. Nos.
5,208,020, 5,416,064 and European Patent EP 0 425 235 B1, the disclosures
of which are hereby expressly incorporated by reference. Liu et al.,
Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates
comprising a maytansinoid designated DM1 linked to the monoclonal
antibody C242 directed against human colorectal cancer. The conjugate was
found to be highly cytotoxic towards cultured colon cancer cells, and
showed antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in which a
maytansinoid was conjugated via a disulfide linker to the murine antibody
A7 binding to an antigen on human colon cancer cell lines, or to another
murine monoclonal antibody TA.1 that binds the HER-2/neu oncogene. The
cytotoxicity of the TA.1-maytansinoid conjugate was tested in vitro on
the human breast cancer cell line SK-BR-3, which expresses
3×105 HER-2 surface antigens per cell. The drug conjugate
achieved a degree of cytotoxicity similar to the free maytansinoid drug,
which could be increased by increasing the number of maytansinoid
molecules per antibody molecule. The A7-maytansinoid conjugate showed low
systemic cytotoxicity in mice.

[0360]Antibody-maytansinoid conjugates are prepared by chemically linking
an antibody to a maytansinoid molecule without significantly diminishing
the biological activity of either the antibody or the maytansinoid
molecule. See, e.g., U.S. Pat. No. 5,208,020 (the disclosure of which is
hereby expressly incorporated by reference). An average of 3-4
maytansinoid molecules conjugated per antibody molecule has shown
efficacy in enhancing cytotoxicity of target cells without negatively
affecting the function or solubility of the antibody, although even one
molecule of toxin/antibody would be expected to enhance cytotoxicity over
the use of naked antibody. Maytansinoids are well known in the art and
can be synthesized by known techniques or isolated from natural sources.
Suitable maytansinoids are disclosed, for example, in U.S. Pat. No.
5,208,020 and in the other patents and nonpatent publications referred to
hereinabove. Preferred maytansinoids are maytansinol and maytansinol
analogues modified in the aromatic ring or at other positions of the
maytansinol molecule, such as various maytansinol esters.

[0361]There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those disclosed
in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, Chari et al.,
Cancer Research 52:127-131 (1992), and U.S. patent application Ser. No.
10/960,602, filed Oct. 8, 2004, the disclosures of which are hereby
expressly incorporated by reference. Antibody-maytansinoid conjugates
comprising the linker component SMCC may be prepared as disclosed in U.S.
patent application Ser. No. 10/960,602, filed Oct. 8, 2004. The linking
groups include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile groups,
as disclosed in the above-identified patents, disulfide and thioether
groups being preferred. Additional linking groups are described and
exemplified herein.

[0363]The linker may be attached to the maytansinoid molecule at various
positions, depending on the type of the link For example, an ester
linkage may be formed by reaction with a hydroxyl group using
conventional coupling techniques. The reaction may occur at the C-3
position having a hydroxyl group, the C-14 position modified with
hydroxymethyl, the C-15 position modified with a hydroxyl group, and the
C-20 position having a hydroxyl group. In a preferred embodiment, the
linkage is formed at the C-3 position of maytansinol or a maytansinol
analogue.

[0364]ii. Auristatins and Dolastatins

[0365]In some embodiments, the immunoconjugate comprises an antibody of
the invention conjugated to dolastatins or dolostatin peptidic analogs
and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).
Dolastatins and auristatins have been shown to interfere with microtubule
dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al
(2001) Antimicrob. Agents and Chemother. 45(12):3580-3584) and have
anticancer (U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et
al (1998) Antimicrob. Agents Chemother. 42:2961-2965). The dolastatin or
auristatin drug moiety may be attached to the antibody through the N
(amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).

[0366]Exemplary auristatin embodiments include the N-terminus linked
monomethylauristatin drug moieties DE and DF, disclosed in
"Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Ser.
No. 10/983,340, filed Nov. 5, 2004, the disclosure of which is expressly
incorporated by reference in its entirety.

[0369]In other embodiments, the immunoconjugate comprises an antibody of
the invention conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S. Pat. Nos.
5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,
5,773,001, 5,877,296 (all to American Cyanamid Company). Structural
analogues of calicheamicin which may be used include, but are not limited
to, γ1I, α2I, α3I,
N-acetyl-γ1I, PSAG and θI1 (Hinman et
al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research
58:2925-2928 (1998) and the aforementioned U.S. patents to American
Cyanamid). Another anti-tumor drug that the antibody can be conjugated is
QFA which is an antifolate. Both calicheamicin and QFA have intracellular
sites of action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated internalization
greatly enhances their cytotoxic effects.

[0370]iv. Other Cytotoxic Agents

[0371]Other antitumor agents that can be conjugated to the antibodies of
the invention include BCNU, streptozoicin, vincristine and
5-fluorouracil, the family of agents known collectively LL-E33288 complex
described in U.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins
(U.S. Pat. No. 5,877,296).

[0373]The present invention further contemplates an immunoconjugate formed
between an antibody and a compound with nucleolytic activity (e.g., a
ribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

[0374]For selective destruction of the tumor, the antibody may comprise a
highly radioactive atom. A variety of radioactive isotopes are available
for the production of radioconjugated antibodies. Examples include
At211, I131, I125, Y90, Re186, Re188,
Sm153, Bi212, P32, Pb212 and radioactive isotopes of
Lu. When the conjugate is used for detection, it may comprise a
radioactive atom for scintigraphic studies, for example tc99m or
I123, or a spin label for nuclear magnetic resonance (NMR) imaging
(also known as magnetic resonance imaging, mri), such as iodine-123
again, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15,
oxygen-17, gadolinium, manganese or iron.

[0375]The radio- or other labels may be incorporated in the conjugate in
known ways. For example, the peptide may be biosynthesized or may be
synthesized by chemical amino acid synthesis using suitable amino acid
precursors involving, for example, fluorine-19 in place of hydrogen.
Labels such as tc99m or I123, Re186, Re188 and
In111 can be attached via a cysteine residue in the peptide.
Yttrium-90 can be attached via a lysine residue. The IODOGEN method
(Fraker et al (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be
used to incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in
detail.

[0376]Conjugates of the antibody and cytotoxic agent may be made using a
variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such as
dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such
as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such
as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as
1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be
prepared as described in Vitetta et al., Science 238:1098 (1987).
Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene
triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for
conjugation of radionucleotide to the antibody. See WO94/11026. The
linker may be a "cleavable linker" facilitating release of the cytotoxic
drug in the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.

[0379]In the antibody drug conjugates (ADC) of the invention, an antibody
(Ab) is conjugated to one or more drug moieties (D), e.g. about 1 to
about 20 drug moieties per antibody, through a linker (L). The ADC of
Formula I may be prepared by several routes, employing organic chemistry
reactions, conditions, and reagents known to those skilled in the art,
including: (1) reaction of a nucleophilic group of an antibody with a
bivalent linker reagent, to form Ab-L, via a covalent bond, followed by
reaction with a drug moiety D; and (2) reaction of a nucleophilic group
of a drug moiety with a bivalent linker reagent, to form D-L, via a
covalent bond, followed by reaction with the nucleophilic group of an
antibody. Additional methods for preparing ADC are described herein.

Ab-(L-D)p I

[0380]The linker may be composed of one or more linker components.
Exemplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"),
N-Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"), N-Succinimidyl
4-(N-maleimidomethyl)cyclohexane-1 carboxylate ("SMCC"), and
N-Succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). Additional linker
components are known in the art and some are described herein. See also
"Monomethylvaline Compounds Capable of Conjugation to Ligands", U.S. Ser.
No. 10/983,340, filed Nov. 5, 2004, the contents of which are hereby
incorporated by reference in its entirety.

[0381]In some embodiments, the linker may comprise amino acid residues.
Exemplary amino acid linker components include a dipeptide, a tripeptide,
a tetrapeptide or a pentapeptide. Exemplary dipeptides include:
valine-citrulline (vc or val-cit), alanine-phenylalanine (af or ala-phe).
Exemplary tripeptides include: glycine-valine-citrulline (gly-val-cit)
and glycine-glycine-glycine (gly-gly-gly). Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring amino
acid analogs, such as citrulline Amino acid linker components can be
designed and optimized in their selectivity for enzymatic cleavage by a
particular enzymes, for example, a tumor-associated protease, cathepsin
B, C and D, or a plasmin protease.

[0382]Nucleophilic groups on antibodies include, but are not limited to:
(i) N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,
(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or
amino groups where the antibody is glycosylated. Amine, thiol, and
hydroxyl groups are nucleophilic and capable of reacting to form covalent
bonds with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such as
haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.
Certain antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made reactive for conjugation with linker
reagents by treatment with a reducing agent such as DTT (dithiothreitol).
Each cysteine bridge will thus form, theoretically, two reactive thiol
nucleophiles. Additional nucleophilic groups can be introduced into
antibodies through the reaction of lysines with 2-iminothiolane (Traut's
reagent) resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three, four, or more cysteine residues (e.g.,
preparing mutant antibodies comprising one or more non-native cysteine
amino acid residues).

[0383]Antibody drug conjugates of the invention may also be produced by
modification of the antibody to introduce electrophilic moieties, which
can react with nucleophilic substituents on the linker reagent or drug.
The sugars of glycosylated antibodies may be oxidized, e.g. with
periodate oxidizing reagents, to form aldehyde or ketone groups which may
react with the amine group of linker reagents or drug moieties. The
resulting imine Schiff base groups may form a stable linkage, or may be
reduced, e.g. by borohydride reagents to form stable amine linkages. In
one embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either glactose oxidase or sodium meta-periodate may yield
carbonyl (aldehyde and ketone) groups in the protein that can react with
appropriate groups on the drug (Hermanson, Bioconjugate Techniques). In
another embodiment, proteins containing N-terminal serine or threonine
residues can react with sodium meta-periodate, resulting in production of
an aldehyde in place of the first amino acid (Geoghegan & Stroh, (1992)
Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such aldehyde can
be reacted with a drug moiety or linker nucleophile.

[0385]Alternatively, a fusion protein comprising the antibody and
cytotoxic agent may be made, e.g., by recombinant techniques or peptide
synthesis. The length of DNA may comprise respective regions encoding the
two portions of the conjugate either adjacent one another or separated by
a region encoding a linker peptide which does not destroy the desired
properties of the conjugate.

[0386]In yet another embodiment, the antibody may be conjugated to a
"receptor" (such streptavidin) for utilization in tumor pre-targeting
wherein the antibody-receptor conjugate is administered to the patient,
followed by removal of unbound conjugate from the circulation using a
clearing agent and then administration of a "ligand" (e.g., avidin) which
is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Pharmaceutical Formulations

[0387]Therapeutic formulations comprising an antibody of the invention are
prepared for storage by mixing the antibody having the desired degree of
purity with optional physiologically acceptable carriers, excipients or
stabilizers (Remington: The Science and Practice of Pharmacy 20th edition
(2000)), in the form of aqueous solutions, lyophilized or other dried
formulations. Acceptable carriers, excipients, or stabilizers are
nontoxic to recipients at the dosages and concentrations employed, and
include buffers such as phosphate, citrate, histidine and other organic
acids; antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides; proteins,
such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose, or
dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants such as TWEEN®, PLURONICS® or polyethylene glycol
(PEG).

[0388]The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not adversely
affect each other. Such molecules are suitably present in combination in
amounts that are effective for the purpose intended.

[0389]The active ingredients may also be entrapped in microcapsule
prepared, for example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsule and poly-(methylmethacylate) microcapsule,
respectively, in colloidal drug delivery systems (for example, liposomes,
albumin microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington: The
Science and Practice of Pharmacy 20th edition (2000).

[0390]The formulations to be used for in vivo administration must be
sterile. This is readily accomplished by filtration through sterile
filtration membranes.

[0391]Sustained-release preparations may be prepared. Suitable examples of
sustained-release preparations include semipermeable matrices of solid
hydrophobic polymers containing the immunoglobulin of the invention,
which matrices are in the form of shaped articles, e.g., films, or
microcapsule. Examples of sustained-release matrices include polyesters,
hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or
poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers
of L-glutamic acid and γ ethyl-L-glutamate, non-degradable
ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers
such as the LUPRON DEPOT® (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinyl
acetate and lactic acid-glycolic acid enable release of molecules for
over 100 days, certain hydrogels release proteins for shorter time
periods. When encapsulated immunoglobulins remain in the body for a long
time, they may denature or aggregate as a result of exposure to moisture
at 37° C., resulting in a loss of biological activity and possible
changes in immunogenicity. Rational strategies can be devised for
stabilization depending on the mechanism involved. For example, if the
aggregation mechanism is discovered to be intermolecular S--S bond
formation through thio-disulfide interchange, stabilization may be
achieved by modifying sulfhydryl residues, lyophilizing from acidic
solutions, controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.

Uses

[0392]An antibody of the present invention may be used in, for example, in
vitro, ex vivo and in vivo therapeutic methods.

[0393]In one aspect, the invention provides methods for treating or
preventing a tumor, a cancer, and/or a cell proliferative disorder
associated with increased expression and/or activity of DLL4, the methods
comprising administering an effective amount of an anti-DLL4 antibody to
a subject in need of such treatment.

[0394]In one aspect, the invention provides methods for reducing,
inhibiting, blocking, or preventing growth of a tumor or cancer, the
methods comprising administering an effective amount of an anti-DLL4
antibody to a subject in need of such treatment.

[0395]In one aspect, the invention provides methods for treating a tumor,
a cancer, and/or a cell proliferative disorder comprising administering
an effective amount of an anti-DLL4 antibody to a subject in need of such
treatment.

[0396]In one aspect, the invention provides methods for inhibiting
angiogenesis comprising administering an effective amount of an anti-DLL4
antibody to a subject in need of such treatment.

[0397]In one aspect, the invention provides methods for treating a
pathological condition associated with angiogenesis comprising
administering an effective amount of an anti-DLL4 antibody to a subject
in need of such treatment. In some embodiments, the pathological
condition associated with angiogenesis is a tumor, a cancer, and/or a
cell proliferative disorder. In some embodiments, the pathological
condition associated with angiogenesis is an intraocular neovascular
disease.

[0398]Moreover, at least some of the antibodies of the invention can bind
antigen from other species. Accordingly, the antibodies of the invention
can be used to bind specific antigen activity, e.g., in a cell culture
containing the antigen, in human subjects or in other mammalian subjects
having the antigen with which an antibody of the invention cross-reacts
(e.g. chimpanzee, baboon, marmoset, cynomolgus and rhesus, pig or mouse).
In one embodiment, the antibody of the invention can be used for
inhibiting antigen activities by contacting the antibody with the antigen
such that antigen activity is inhibited. Preferably, the antigen is a
human protein molecule.

[0399]In one embodiment, an antibody of the invention can be used in a
method for binding an antigen in a subject suffering from a disorder
associated with increased antigen expression and/or activity, comprising
administering to the subject an antibody of the invention such that the
antigen in the subject is bound. Preferably, the antigen is a human
protein molecule and the subject is a human subject. Alternatively, the
subject can be a mammal expressing the antigen with which an antibody of
the invention binds. Still further the subject can be a mammal into which
the antigen has been introduced (e.g., by administration of the antigen
or by expression of an antigen transgene). An antibody of the invention
can be administered to a human subject for therapeutic purposes.
Moreover, an antibody of the invention can be administered to a non-human
mammal expressing an antigen with which the immunoglobulin cross-reacts
(e.g., a primate, pig or mouse) for veterinary purposes or as an animal
model of human disease. Regarding the latter, such animal models may be
useful for evaluating the therapeutic efficacy of antibodies of the
invention (e.g., testing of dosages and time courses of administration).

[0400]The antibodies of the invention can be used to treat, inhibit, delay
progression of, prevent/delay recurrence of, ameliorate, or prevent
diseases, disorders or conditions associated with expression and/or
activity of one or more antigen molecules.

[0402]In certain embodiments, an immunoconjugate comprising an antibody
conjugated with one or more cytotoxic agent(s) is administered to the
patient. In some embodiments, the immunoconjugate and/or antigen to which
it is bound is/are internalized by the cell, resulting in increased
therapeutic efficacy of the immunoconjugate in killing the target cell to
which it binds. In one embodiment, the cytotoxic agent targets or
interferes with nucleic acid in the target cell. In one embodiment, the
cytotoxic agent targets or interferes with microtubule polymerization.
Examples of such cytotoxic agents include any of the chemotherapeutic
agents noted herein (such as a maytansinoid, auristatin, dolastatin, or a
calicheamicin), a radioactive isotope, or a ribonuclease or a DNA
endonuclease.

[0403]Antibodies of the invention can be used either alone or in
combination with other compositions in a therapy. For instance, an
antibody of the invention may be co-administered with another antibody,
chemotherapeutic agent(s) (including cocktails of chemotherapeutic
agents), other cytotoxic agent(s), anti-angiogenic agent(s), cytokines,
and/or growth inhibitory agent(s). Where an antibody of the invention
inhibits tumor growth, it may be particularly desirable to combine it
with one or more other therapeutic agent(s) which also inhibits tumor
growth, e.g. anti-VEGF agents including antibodies to VEGF.
Alternatively, or additionally, the patient may receive combined
radiation therapy (e.g. external beam irradiation or therapy with a
radioactive labeled agent, such as an antibody). Such combined therapies
noted above include combined administration (where the two or more agents
are included in the same or separate formulations), and separate
administration, in which case, administration of the antibody of the
invention can occur prior to, and/or following, administration of the
adjunct therapy or therapies.

[0404]Combination Therapies

[0405]As indicated above, the invention provides combined therapies in
which an anti-DLL4 antibody is administered with another therapy. For
example, anti-DLL4 antibodies are used in combinations with anti-cancer
therapeutics or anti-neovascularization therapeutics to treat various
neoplastic or non-neoplastic conditions. In one embodiment, the
neoplastic or non-neoplastic condition is characterized by pathological
disorder associated with aberrant or undesired angiogenesis. The
anti-DLL4 antibody can be administered serially or in combination with
another agent that is effective for those purposes, either in the same
composition or as separate compositions. Alternatively, or additionally,
multiple inhibitors of DLL4 can be administered.

[0406]The administration of the anti-DLL4 antibody can be done
simultaneously, e.g., as a single composition or as two or more distinct
compositions using the same or different administration routes.
Alternatively, or additionally, the administration can be done
sequentially, in any order. In certain embodiments, intervals ranging
from minutes to days, to weeks to months, can be present between the
administrations of the two or more compositions. For example, the
anti-cancer agent may be administered first, followed by the DLL4
inhibitor. However, simultaneous administration or administration of the
anti-DLL4 antibody first is also contemplated.

[0407]The effective amounts of therapeutic agents administered in
combination with an anti-DLL4 antibody will be at the physician's or
veterinarian's discretion. Dosage administration and adjustment is done
to achieve maximal management of the conditions to be treated. The dose
will additionally depend on such factors as the type of therapeutic agent
to be used and the specific patient being treated. Suitable dosages for
the anti-cancer agent are those presently used and can be lowered due to
the combined action (synergy) of the anti-cancer agent and the anti-DLL4
antibody. In certain embodiments, the combination of the inhibitors
potentiates the efficacy of a single inhibitor. The term "potentiate"
refers to an improvement in the efficacy of a therapeutic agent at its
common or approved dose. See also the section entitled Pharmaceutical
Compositions herein.

[0408]Typically, the anti-DLL4 antibodies and anti-cancer agents are
suitable for the same or similar diseases to block or reduce a
pathological disorder such as tumor growth or growth of a cancer cell. In
one embodiment the anti-cancer agent is an anti-angiogenesis agent.

[0409]Antiangiogenic therapy in relationship to cancer is a cancer
treatment strategy aimed at inhibiting the development of tumor blood
vessels required for providing nutrients to support tumor growth. Because
angiogenesis is involved in both primary tumor growth and metastasis, the
antiangiogenic treatment provided by the invention is capable of
inhibiting the neoplastic growth of tumor at the primary site as well as
preventing metastasis of tumors at the secondary sites, therefore
allowing attack of the tumors by other therapeutics.

[0410]Many anti-angiogenic agents have been identified and are known in
the arts, including those listed herein, e.g., listed under Definitions,
and by, e.g., Carmeliet and Jain, Nature 407:249-257 (2000); Ferrara et
al., Nature Reviews: Drug Discovery, 3:391-400 (2004); and Sato Int. J.
Clin. Oncol., 8:200-206 (2003). See also, US Patent Application
US20030055006. In one embodiment, an anti-DLL4 antibody is used in
combination with an anti-VEGF neutralizing antibody (or fragment) and/or
another VEGF antagonist or a VEGF receptor antagonist including, but not
limited to, for example, soluble VEGF receptor (e.g., VEGFR-1, VEGFR-2,
VEGFR-3, neuropillins (e.g., NRP1, NRP2)) fragments, aptamers capable of
blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low molecule
weight inhibitors of VEGFR tyrosine kinases (RTK), antisense strategies
for VEGF, ribozymes against VEGF or VEGF receptors, antagonist variants
of VEGF; and any combinations thereof. Alternatively, or additionally,
two or more angiogenesis inhibitors may optionally be co-administered to
the patient in addition to VEGF antagonist and other agent. In certain
embodiment, one or more additional therapeutic agents, e.g., anti-cancer
agents, can be administered in combination with anti-DLL4 antibody, the
VEGF antagonist, and an anti-angiogenesis agent.

[0411]In certain aspects of the invention, other therapeutic agents useful
for combination tumor therapy with a anti-DLL4 antibody include other
cancer therapies, (e.g., surgery, radiological treatments (e.g.,
involving irradiation or administration of radioactive substances),
chemotherapy, treatment with anti-cancer agents listed herein and known
in the art, or combinations thereof). Alternatively, or additionally, two
or more antibodies binding the same or two or more different antigens
disclosed herein can be co-administered to the patient. Sometimes, it may
be beneficial to also administer one or more cytokines to the patient.

[0412]Chemotherapeutic Agents

[0413]In certain aspects, the invention provides a method of blocking or
reducing tumor growth or growth of a cancer cell, by administering
effective amounts of an antagonist of DLL4 and/or an angiogenesis
inhibitor(s) and one or more chemotherapeutic agents to a patient
susceptible to, or diagnosed with, cancer. A variety of chemotherapeutic
agents may be used in the combined treatment methods of the invention. An
exemplary and non-limiting list of chemotherapeutic agents contemplated
is provided herein under "Definitions."

[0414]As will be understood by those of ordinary skill in the art, the
appropriate doses of chemotherapeutic agents will be generally around
those already employed in clinical therapies wherein the
chemotherapeutics are administered alone or in combination with other
chemotherapeutics. Variation in dosage will likely occur depending on the
condition being treated. The physician administering treatment will be
able to determine the appropriate dose for the individual subject.

[0415]The invention also provides methods and compositions for inhibiting
or preventing relapse tumor growth or relapse cancer cell growth. Relapse
tumor growth or relapse cancer cell growth is used to describe a
condition in which patients undergoing or treated with one or more
currently available therapies (e.g., cancer therapies, such as
chemotherapy, radiation therapy, surgery, hormonal therapy and/or
biological therapy/immunotherapy, anti-VEGF antibody therapy,
particularly a standard therapeutic regimen for the particular cancer) is
not clinically adequate to treat the patients or the patients are no
longer receiving any beneficial effect from the therapy such that these
patients need additional effective therapy. As used herein, the phrase
can also refer to a condition of the "non-responsive/refractory" patient,
e.g., which describe patients who respond to therapy yet suffer from side
effects, develop resistance, do not respond to the therapy, do not
respond satisfactorily to the therapy, etc. In various embodiments, a
cancer is relapse tumor growth or relapse cancer cell growth where the
number of cancer cells has not been significantly reduced, or has
increased, or tumor size has not been significantly reduced, or has
increased, or fails any further reduction in size or in number of cancer
cells. The determination of whether the cancer cells are relapse tumor
growth or relapse cancer cell growth can be made either in vivo or in
vitro by any method known in the art for assaying the effectiveness of
treatment on cancer cells, using the art-accepted meanings of "relapse"
or "refractory" or "non-responsive" in such a context. A tumor resistant
to anti-VEGF treatment is an example of a relapse tumor growth.

[0416]The invention provides methods of blocking or reducing relapse tumor
growth or relapse cancer cell growth in a subject by administering one or
more anti-DLL4 antibodies to block or reduce the relapse tumor growth or
relapse cancer cell growth in subject. In certain embodiments, the
antagonist can be administered subsequent to the cancer therapeutic. In
certain embodiments, the anti-DLL4 antibodies are administered
simultaneously with cancer therapy. Alternatively, or additionally, the
anti-DLL4 antibody therapy alternates with another cancer therapy, which
can be performed in any order. The invention also encompasses methods for
administering one or more inhibitory antibodies to prevent the onset or
recurrence of cancer in patients predisposed to having cancer. Generally,
the subject was or is concurrently undergoing cancer therapy. In one
embodiment, the cancer therapy is treatment with an anti-angiogenesis
agent, e.g., a VEGF antagonist. The anti-angiogenesis agent includes
those known in the art and those found under the Definitions herein. In
one embodiment, the anti-angiogenesis agent is an anti-VEGF neutralizing
antibody or fragment (e.g. AVASTIN® (Genentech, South San Francisco,
Calif.) or LUCENTIS® (Genentech, South San Francisco, Calif.)),
Y0317, M4, G6, B20, 2C3, etc.). See, e.g., U.S. Pat. Nos. 6,582,959,
6,884,879, 6,703,020; WO98/45332; WO 96/30046; WO94/10202; EP 0666868B1;
US Patent Applications 20030206899, 20030190317, 20030203409, and
20050112126; Popkov et al., Journal of Immunological Methods 288:149-164
(2004); and, WO2005012359. Additional agents can be administered in
combination with VEGF antagonist and an anti-DLL4 antibody for blocking
or reducing relapse tumor growth or relapse cancer cell growth, e.g., see
section entitled Combination Therapies herein.

[0417]The antibody of the invention (and adjunct therapeutic agent) is/are
administered by any suitable means, including parenteral, subcutaneous,
intraperitoneal, intrapulmonary, and intranasal, and, if desired for
local treatment, intralesional or intravitreal administration. Parenteral
infusions include intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous administration. In addition, the
antibody is suitably administered by pulse infusion, particularly with
declining doses of the antibody. Dosing can be by any suitable route,
e.g. by injections, such as intravenous or subcutaneous injections,
depending in part on whether the administration is brief or chronic.

[0418]The antibody composition of the invention will be formulated, dosed,
and administered in a fashion consistent with good medical practice.
Factors for consideration in this context include the particular disorder
being treated, the particular mammal being treated, the clinical
condition of the individual patient, the cause of the disorder, the site
of delivery of the agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners. The
antibody need not be, but is optionally formulated with one or more
agents currently used to prevent or treat the disorder in question. The
effective amount of such other agents depends on the amount of antibodies
of the invention present in the formulation, the type of disorder or
treatment, and other factors discussed above. These are generally used in
the same dosages and with administration routes as used hereinbefore or
about from 1 to 99% of the heretofore employed dosages.

[0419]For the prevention or treatment of disease, the appropriate dosage
of an antibody of the invention (when used alone or in combination with
other agents such as chemotherapeutic agents) will depend on the type of
disease to be treated, the type of antibody, the severity and course of
the disease, whether the antibody is administered for preventive or
therapeutic purposes, previous therapy, the patient's clinical history
and response to the antibody, and the discretion of the attending
physician. The antibody is suitably administered to the patient at one
time or over a series of treatments. Depending on the type and severity
of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of
antibody is an initial candidate dosage for administration to the
patient, whether, for example, by one or more separate administrations,
or by continuous infusion. One typical daily dosage might range from
about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned
above. For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. One exemplary dosage of the
antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.
Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10
mg/kg (or any combination thereof) may be administered to the patient.
Such doses may be administered intermittently, e.g. every week or every
three weeks (e.g. such that the patient receives from about two to about
twenty, e.g. about six doses of the antibody). An initial higher loading
dose, followed by one or more lower doses may be administered. An
exemplary dosing regimen comprises administering an initial loading dose
of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg
of the antibody. However, other dosage regimens may be useful. The
progress of this therapy is easily monitored by conventional techniques
and assays.

[0420]The anti-DLL4 antibodies of the invention are useful in assays
detecting DLL4 expression (such as diagnostic or prognostic assays) in
specific cells or tissues wherein the antibodies are labeled as described
below and/or are immobilized on an insoluble matrix.

[0421]In another aspect, the invention provides methods for detection of
DLL4, the methods comprising detecting DLL4-anti-DLL4 antibody complex in
the sample. The term "detection" as used herein includes qualitative
and/or quantitative detection (measuring levels) with or without
reference to a control.

[0422]In another aspect, the invention provides methods for diagnosing a
disorder associated with DLL4 expression and/or activity, the methods
comprising detecting DLL4-anti-DLL4 antibody complex in a biological
sample from a patient having or suspected of having the disorder. In some
embodiments, the DLL4 expression is increased expression or abnormal
(undesired) expression. In some embodiments, the disorder is a tumor,
cancer, and/or a cell proliferative disorder.

[0423]In another aspect, the invention provides any of the anti-DLL4
antibodies described herein, wherein the anti-DLL4 antibody comprises a
detectable label.

[0424]In another aspect, the invention provides a complex of any of the
anti-DLL4 antibodies described herein and DLL4. In some embodiments, the
complex is in vivo or in vitro. In some embodiments, the complex
comprises a cancer cell. In some embodiments, the anti-DLL4 antibody is
detectably labeled.

[0425]Anti-DLL4 antibodies can be used for the detection of DLL4 in any
one of a number of well known detection assay methods. For example, a
biological sample may be assayed for DLL4 by obtaining the sample from a
desired source, admixing the sample with anti-DLL4 antibody to allow the
antibody to form antibody/DLL4 complex with any DLL4 present in the
mixture, and detecting any antibody/DLL4 complex present in the mixture.
The biological sample may be prepared for assay by methods known in the
art which are suitable for the particular sample. The methods of admixing
the sample with antibodies and the methods of detecting antibody/DLL4
complex are chosen according to the type of assay used. Such assays
include immunohistochemistry, competitive and sandwich assays, and steric
inhibition assays.

[0426]Analytical methods for DLL4 all use one or more of the following
reagents: labeled DLL4 analogue, immobilized DLL4 analogue, labeled
anti-DLL4 antibody, immobilized anti-DLL4 antibody and steric conjugates.
The labeled reagents also are known as "tracers."

[0427]The label used is any detectable functionality that does not
interfere with the binding of DLL4 and anti-DLL4 antibody. Numerous
labels are known for use in immunoassay, examples including moieties that
may be detected directly, such as fluorochrome, chemiluminescent, and
radioactive labels, as well as moieties, such as enzymes, that must be
reacted or derivatized to be detected. Examples of such labels include:
The label used is any detectable functionality that does not interfere
with the binding of DLL4 and anti-DLL4 antibody. Numerous labels are
known for use in immunoassay, examples including moieties that may be
detected directly, such as fluorochrome, chemiluminescent, and
radioactive labels, as well as moieties, such as enzymes, that must be
reacted or derivatized to be detected. Examples of such labels include
the radioisotopes 32P, 14C, 125I, 3H, and 131I,
fluorophores such as rare earth chelates or fluorescein and its
derivatives, rhodamine and its derivatives, dansyl, umbelliferone,
luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.
Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish
peroxidase (HRP), alkaline phosphatase, β-galactosidase,
glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,
galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic
oxidases such as uricase and xanthine oxidase, coupled with an enzyme
that employs hydrogen peroxide to oxidize a dye precursor such as HRP,
lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,
bacteriophage labels, stable free radicals, and the like.

[0428]Conventional methods are available to bind these labels covalently
to proteins or polypeptides. For instance, coupling agents such as
dialdehydes, carbodiimides, dimaleimides, bis-imidates, bis-diazotized
benzidine, and the like may be used to tag the antibodies with the
above-described fluorescent, chemiluminescent, and enzyme labels. See,
for example, U.S. Pat. Nos. 3,940,475 (fluorimetry) and 3,645,090
(enzymes); Hunter et al., Nature, 144: 945 (1962); David et al.,
Biochemistry, 13: 1014-1021 (1974); Pain et al., J. Immunol. Methods, 40:
219-230 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407-412
(1982). Preferred labels herein are enzymes such as horseradish
peroxidase and alkaline phosphatase. The conjugation of such label,
including the enzymes, to the antibody is a standard manipulative
procedure for one of ordinary skill in immunoassay techniques. See, for
example, O'Sullivan et al., "Methods for the Preparation of
Enzyme-antibody Conjugates for Use in Enzyme Immunoassay," in Methods in
Enzymology, ed. J. J. Langone and H. Van Vunakis, Vol. 73 (Academic
Press, New York, N.Y., 1981), pp. 147-166.

[0429]Immobilization of reagents is required for certain assay methods.
Immobilization entails separating the anti-DLL4 antibody from any DLL4
that remains free in solution. This conventionally is accomplished by
either insolubilizing the anti-DLL4 antibody or DLL4 analogue before the
assay procedure, as by adsorption to a water-insoluble matrix or surface
(Bennich et al., U.S. Pat. No. 3,720,760), by covalent coupling (for
example, using glutaraldehyde cross-linking), or by insolubilizing the
anti-DLL4 antibody or DLL4 analogue afterward, e.g., by
immunoprecipitation.

[0430]The expression of proteins in a sample may be examined using
immunohistochemistry and staining protocols Immunohistochemical staining
of tissue sections has been shown to be a reliable method of assessing or
detecting presence of proteins in a sample Immunohistochemistry ("IHC")
techniques utilize an antibody to probe and visualize cellular antigens
in situ, generally by chromogenic or fluorescent methods. For sample
preparation, a tissue or cell sample from a mammal (typically a human
patient) may be used. Examples of samples include, but are not limited
to, cancer cells such as colon, breast, prostate, ovary, lung, stomach,
pancreas, lymphoma, and leukemia cancer cells. The sample can be obtained
by a variety of procedures known in the art including, but not limited to
surgical excision, aspiration or biopsy. The tissue may be fresh or
frozen. In one embodiment, the sample is fixed and embedded in paraffin
or the like. The tissue sample may be fixed (i.e. preserved) by
conventional methodology. One of ordinary skill in the art will
appreciate that the choice of a fixative is determined by the purpose for
which the sample is to be histologically stained or otherwise analyzed.
One of ordinary skill in the art will also appreciate that the length of
fixation depends upon the size of the tissue sample and the fixative
used.

[0431]IHC may be performed in combination with additional techniques such
as morphological staining and/or fluorescence in-situ hybridization. Two
general methods of IHC are available; direct and indirect assays.
According to the first assay, binding of antibody to the target antigen
(e.g., DLL4) is determined directly. This direct assay uses a labeled
reagent, such as a fluorescent tag or an enzyme-labeled primary antibody,
which can be visualized without further antibody interaction. In a
typical indirect assay, unconjugated primary antibody binds to the
antigen and then a labeled secondary antibody binds to the primary
antibody. Where the secondary antibody is conjugated to an enzymatic
label, a chromogenic or fluorogenic substrate is added to provide
visualization of the antigen. Signal amplification occurs because several
secondary antibodies may react with different epitopes on the primary
antibody.

[0432]The primary and/or secondary antibody used for immunohistochemistry
typically will be labeled with a detectable moiety. Numerous labels are
available which can be generally grouped into the following categories:

[0433]Aside from the sample preparation procedures discussed above,
further treatment of the tissue section prior to, during or following IHC
may be desired, For example, epitope retrieval methods, such as heating
the tissue sample in citrate buffer may be carried out (see, e.g., Leong
et al. Appi. Immunohistochem. 4(3):201 (1996)).

[0434]Following an optional blocking step, the tissue section is exposed
to primary antibody for a sufficient period of time and under suitable
conditions such that the primary antibody binds to the target protein
antigen in the tissue sample. Appropriate conditions for achieving this
can be determined by routine experimentation. The extent of binding of
antibody to the sample is determined by using any one of the detectable
labels discussed above. Preferably, the label is an enzymatic label (e.g.
HRPO) which catalyzes a chemical alteration of the chromogenic substrate
such as 3,3'-diaminobenzidine chromogen. Preferably the enzymatic label
is conjugated to antibody which binds specifically to the primary
antibody (e.g. the primary antibody is rabbit polyclonal antibody and
secondary antibody is goat anti-rabbit antibody).

[0435]Specimens thus prepared may be mounted and coverslipped. Slide
evaluation is then determined, e.g. using a microscope, and staining
intensity criteria, routinely used in the art, may be employed. Staining
intensity criteria may be evaluated as follows:

TABLE-US-00007
TABLE 2
Staining Pattern Score
No staining is observed in cells. 0
Faint/barely perceptible staining is detected in more 1+
than 10% of the cells.
Weak to moderate staining is observed in more than 10% 2+
of the cells.
Moderate to strong staining is observed in more than 3+
10% of the cells.

[0436]Typically, a staining pattern score of about 2+ or higher in an IHC
assay is diagnostic and/or prognostic. In some embodiments, a staining
pattern score of about 1+ or higher is diagnostic and/or prognostic. In
other embodiments, a staining pattern score of about 3 of higher is
diagnostic and/or prognostic. It is understood that when cells and/or
tissue from a tumor or colon adenoma are examined using IHC, staining is
generally determined or assessed in tumor cell and/or tissue (as opposed
to stromal or surrounding tissue that may be present in the sample).

[0437]Other assay methods, known as competitive or sandwich assays, are
well established and widely used in the commercial diagnostics industry.

[0438]Competitive assays rely on the ability of a tracer DLL4 analogue to
compete with the test sample DLL4 for a limited number of anti-DLL4
antibody antigen-binding sites. The anti-DLL4 antibody generally is
insolubilized before or after the competition and then the tracer and
DLL4 bound to the anti-DLL4 antibody are separated from the unbound
tracer and DLL4. This separation is accomplished by decanting (where the
binding partner was preinsolubilized) or by centrifuging (where the
binding partner was precipitated after the competitive reaction). The
amount of test sample DLL4 is inversely proportional to the amount of
bound tracer as measured by the amount of marker substance. Dose-response
curves with known amounts of DLL4 are prepared and compared with the test
results to quantitatively determine the amount of DLL4 present in the
test sample. These assays are called ELISA systems when enzymes are used
as the detectable markers.

[0439]Another species of competitive assay, called a "homogeneous" assay,
does not require a phase separation. Here, a conjugate of an enzyme with
the DLL4 is prepared and used such that when anti-DLL4 antibody binds to
the DLL4 the presence of the anti-DLL4 antibody modifies the enzyme
activity. In this case, the DLL4 or its immunologically active fragments
are conjugated with a bifunctional organic bridge to an enzyme such as
peroxidase. Conjugates are selected for use with anti-DLL4 antibody so
that binding of the anti-DLL4 antibody inhibits or potentiates the enzyme
activity of the label. This method per se is widely practiced under the
name of EMIT.

[0440]Steric conjugates are used in steric hindrance methods for
homogeneous assay. These conjugates are synthesized by covalently linking
a low-molecular-weight hapten to a small DLL4 fragment so that antibody
to hapten is substantially unable to bind the conjugate at the same time
as anti-DLL4 antibody. Under this assay procedure the DLL4 present in the
test sample will bind anti-DLL4 antibody, thereby allowing anti-hapten to
bind the conjugate, resulting in a change in the character of the
conjugate hapten, e.g., a change in fluorescence when the hapten is a
fluorophore.

[0441]Sandwich assays particularly are useful for the determination of
DLL4 or anti-DLL4 antibodies. In sequential sandwich assays an
immobilized anti-DLL4 antibody is used to adsorb test sample DLL4, the
test sample is removed as by washing, the bound DLL4 is used to adsorb a
second, labeled anti-DLL4 antibody and bound material is then separated
from residual tracer. The amount of bound tracer is directly proportional
to test sample DLL4. In "simultaneous" sandwich assays the test sample is
not separated before adding the labeled anti-DLL4. A sequential sandwich
assay using an anti-DLL4 monoclonal antibody as one antibody and a
polyclonal anti-DLL4 antibody as the other is useful in testing samples
for DLL4.

[0442]The foregoing are merely exemplary detection assays for DLL4. Other
methods now or hereafter developed that use anti-DLL4 antibody for the
determination of DLL4 are included within the scope hereof, including the
bioassays described herein.

Articles of Manufacture

[0443]In another aspect of the invention, an article of manufacture
containing materials useful for the treatment, prevention and/or
diagnosis of the disorders described above is provided. The article of
manufacture comprises a container and a label or package insert on or
associated with the container. Suitable containers include, for example,
bottles, vials, syringes, etc. The containers may be formed from a
variety of materials such as glass or plastic. The container holds a
composition which is by itself or when combined with another
composition(s) effective for treating, preventing and/or diagnosing the
condition and may have a sterile access port (for example the container
may be an intravenous solution bag or a vial having a stopper pierceable
by a hypodermic injection needle). At least one active agent in the
composition is an antibody of the invention. The label or package insert
indicates that the composition is used for treating the condition of
choice, such as cancer. Moreover, the article of manufacture may comprise
(a) a first container with a composition contained therein, wherein the
composition comprises an antibody of the invention; and (b) a second
container with a composition contained therein, wherein the composition
comprises a further therapeutic agent, including, e.g. a chemotherapeutic
agent or an anti-angiogenesis agent, including, e.g., an anti-VEGF
antibody (e.g. bevacizumab). The article of manufacture in this
embodiment of the invention may further comprise a package insert
indicating that the first and second antibody compositions can be used to
treat a particular condition, e.g. cancer. Alternatively, or
additionally, the article of manufacture may further comprise a second
(or third) container comprising a pharmaceutically-acceptable buffer,
such as bacteriostatic water for injection (BWFI), phosphate-buffered
saline, Ringer's solution and dextrose solution. It may further include
other materials desirable from a commercial and user standpoint,
including other buffers, diluents, filters, needles, and syringes.

[0444]The following are examples of the methods and compositions of the
invention. It is understood that various other embodiments may be
practiced, given the general description provided above.

EXAMPLES

[0445]Commercially available reagents referred to in the Examples were
used according to manufacturer's instructions unless otherwise indicated.
The source of those cells identified in the following Examples, and
throughout the specification, by ATCC® accession numbers is the
American Type Culture Collection, Manassas, Va. 20108. References cited
in the Examples are listed following the examples. All references cited
herein are hereby incorporated by reference.

Example 1

Materials and Methods

[0446]The following materials and methods were used in the Examples.

[0447]HUVEC fibrin gel bead assay. Details of the HUVEC fibrin gel bead
assay have been described (Nakatsu, M. N. et al. Microvasc Res 66, 102-12
(2003)). Briefly, Cytodex® 3 beads (Amersham Pharmacia Biotech) were
coated with 350-400 HUVECs per bead. About 200 HUVEC-coated beads were
imbedded in fibrin clot in one well of 12-well tissue culture plate.
8×104 SF cells were plated on top of the clot. Assays were
terminated between day 7 and day 9 for immunostaining and imaging. In
some experiments, HUVEC sprouts were visualized by staining with
Biotin-anti-CD31 (clone WM59, eBioscience) and strepavidin-Cy3. For HUVEC
nuclei staining, fibrin gels were fixed overnight in 2% paraformaldehyde
(PFA), and stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma). For
Ki67 staining, fibrin gels were treated with 10X trypsin-EDTA for 5 min
to remove the top layer SF, neutralized with 10% FBS in PBS, and fixed
overnight in 4% PFA. Fibrin gels were then blocked with 10% goat serum in
PBST for 4 hr, incubated overnight with rabbit anti-mouse Ki67
(Ready-To-Use, clone Sp6, LabVision), followed by secondary detection
with anti-rabbit IgG-Cy3 (Jackson ImmunoResearch). All overnight
incubations were done at 4° C.

[0450]Tumor vascular labeling and immunohistochemistry. Mice were
anesthetized with Isoflurane. FITC-labeled Lycopersicon esculentum Lectin
(150 μg in 150 μl of 0.9% NaCl; Vector Laboratories) was injected
i.v. and allowed to circulate for 5 min before systemic perfusion. The
vasculature was perfused transcardially with 1% PFA in PBS for 3 min.
Tumors were removed and post fixed by immersion in the same fixative for
2 hr, followed by an incubation in 30% sucrose overnight for
cryoprotection, then embedded in OCT. Sections (4 μm thickness) were
stained with anti-mouse CD31 (1:50, BD Pharmingen), followed by Alexa 594
goat anti-rat IgG (1:800, Molecular Probes).

[0454]RNA extraction and Real-time quantitative RT-PCR. Extraction of
total RNA from HUVECs in 2-D culture was done using RNeasy® Mini Kit
(Qiagen) as per instructions of the manufacturer. To extract total RNA
from HUVECs growing in fibrin gels, fibrin gels were treated with
10× trypsin-EDTA (Gibco) for 5 min to remove the top layer
fibroblasts, followed by neutralization with 10% FBS in PBS. The gel
clots were then removed from tissue culture wells and subjected to
centrifugation (10K for 5 min) in microtubes to remove excessive fluid.
The resulting gel "pellets" were lysed with lysis buffer (RNeasy®
Mini Kit), and further processed as with HUVECs in 2-D culture. The
quality of RNA was assessed using RNA 6000 Nano Chips and the Agilent
2100 Bioanalyzer (Agilent Technologies). Real-time quantitative RT-PCR
reactions were done in triplicate using 7500 Real Time PCR System
(Applied Biosystems). Human GAPDH was used as reference gene for
normalization. The expression levels are expressed as the mean (±SEM)
fold mRNA changes relative to control from 3 separate determinations. The
forward and reverse primer and probe sequences for VEGFR2, TGFβ2 and
GAPDH were as follows.

[0455]Synthetic phage antibody libraries were built on a single framework
(humanized anti-ErbB2 antibody, 4D5) by introducing diversity within the
complementarity-determining regions (CDRs) of heavy and light chains
(Lee, C. V. et al. J Mol Biol 340, 1073-93 (2004); Liang, W. C. et al. J
Biol Chem 281, 951-61 (2006)). Plate panning with naive libraries was
performed against His-tagged human DLL4 (amino acid 1-404) immobilized on
MaxiSorp® immunoplates. After four rounds of enrichment, clones were
randomly picked and specific binders were identified using phage ELISA.
The resulting hDLL4 binding clones were further screened with His-tagged
murine DLL4 protein to identify cross-species clones. For each positive
phage clone, variable regions of heavy and light chains were subcloned
into pRK expression vectors that were engineered to express full-length
IgG chains. Heavy chain and light chain constructs were co-transfected
into 293 or CHO cells, and the expressed antibodies were purified from
serum-free medium using protein A affinity column. Purified antibodies
were tested by ELISA for blocking the interaction between DLL4 and rat
Notch1-Fc, and by FACS for binding to stable cell lines expressing either
full-length human DLL4 or murine DLL4. For affinity maturation, phage
libraries with three different combination of CDR loops (CDR-L3, -H1, and
-H2) derived from the initial clone of interest were constructed by soft
randomization strategy so that each selected position was mutated to a
non-wild type residue or maintained as wild type at about 50:50 frequency
(Liang et al., 2006, above). High affinity clones were then identified
through four rounds of solution phase panning against both human and
murine His-tagged DLL4 proteins with progressively increased stringency.

Example 3

Characterization of Anti-DLL4 Antibodies

[0456]To determine the binding affinity of the anti-DLL4 Mabs, surface
plasmon resonance (SRP) measurement with a BIAcore®-3000 was used
(BIAcore, Inc., Piscataway, N.J.). Briefly, carboxymethylated dextran
biosensor chips (CMS, BIAcore Inc.) were activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and
N-hydroxysuccinimide (NHS) according to the supplier's instructions.
Anti-DLL4 antibody was diluted with 10 mM sodium acetate, pH 4.8, into 5
μg/ml before injection at a flow rate of SW/minute to achieve
approximately 500 response units (RU) of coupled antibody. Next, 1M
ethanolamine was injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of either human or murine
DLL4-His molecules (0.7 nM to 500 nM) were injected in PBS with 0.05%
Tween® 20 at 25° C. at a flow rate of 25 W/min Association
rates (kon) and dissociation rates (koff) were calculated using a simple
one-to-one Langmuir binding model (BIAcore Evaluation Software version
3.2). The equilibrium dissociation constant (Kd) was calculated as the
ratio koff/kon. The results of this experiment are shown in Table 3.
Antibody YW26.81 exhibited similar Kd values against human and murine
DLL4 (Kd values of 0.1 nM5 and 0.09 nM, respectively), enabling its
evaluation in mouse models.

[0458]Mab YW26.82 selectively binds to mouse and human DLL4. 96-well Nunc
Maxisorp plates were coated with purified recombinant proteins as
indicated (1 μg/ml). The binding of YW26.82 at indicated
concentrations was measured by ELISA assay. Bound antibodies were
detected with anti-human antibody HRP conjugate using TMB as substrate
and OD 450 nm absorbance measurement. Anti-HER2 and recombinant ErbB2-ECD
were used as assay control (FIG. 11b). The results of this experiment are
shown in FIG. 11b. The Mab YW26.82 bound human and mouse DLL4, and did
not detectably bind to human DLL1 and human JAG1. These results
demonstrated that Mab YW26.82 selectively binds to DLL4.

[0459]FACS analysis of 293 cells transiently transfected with vector, full
length DLL4, Jag1 or DLL1 also demonstrated that Mab YW26.82 selectively
bound to DLL4. As shown in FIG. 11c, significant binding of YW26.82 was
only detected on DLL4 transfected cells (top panel). Significant binding
was not detected on DLL1 or Jag1 transfected cells. Expression of Jag1
and DLL1 was confirmed by the binding of recombinant rat Notch1-Fc
(rrNotch1-Fc, middle panel) and recombinant rat Notch2-Fc (rrNotch2-Fc,
bottom panel), respectively. YW26.82, rrNotch1-Fc or rrNotch2-Fc (R& D
system) were used at 2 μg/ml followed by goat anti-human IgG-PE
(1:500, Jackson ImmunoResearch).

[0460]Competition experiments demonstrated that Mab YW26.82 effectively
and selectively blocked the interaction of Notch with DLL4, but not other
Notch ligands. As shown in FIG. 11d, anti-DLL4 Mab blocked the binding of
DLL4-AP, but not DLL1-AP, to coated rNotch1, with a calculated IC50
of ˜12 nM (left panel). Anti-DLL4 Mab blocked the binding of
DLL4-His, but not Jag1-His, to coated rNotch1, with a calculated IC50 of
˜8 nM (right panel).

[0461]Anti-DLL4 Mab YW26.82 specifically bound to endogenously expressed
DLL4 in Human umbilical vein endothelial cells (HUVECs). FACS analysis of
HUVECs transfected with control or DLL4-specific siRNA. YW26.82 was used
at 2 μg/ml, followed by goat anti-human IgG-PE (1:500, Jackson
ImmunoResearch). The results of this experiment are shown in FIG. 11e.
Binding was observed to untransfected HUVECs (control) and to HUVECs
transfected with a control siRNA. By contrast, binding was significantly
reduced in HUVECs transfected with DLL4 siRNA. These experiments
demonstrated that anti-DLL4 Mab YW26.82 specifically binds to
endogenously expressed DLL4 in HUVEC.

[0465]Notably, the hyperproliferation of ECs resulting from blocking Notch
signaling was still dependent on VEGF. In the 3-D fibrin gel culture,
treatment with anti-VEGF Mab abolished most of the EC sprouting, either
in the presence or absence of DBZ (FIG. 7f), raising the possibility that
the hyperproliferative behavior could be in part due to enhanced VEGF
signaling. Indeed, blocking of Notch by YW26.82 or DBZ resulted in
upregulation of VEGFR2 (FIG. 7g). Conversely, activation of Notch by
immobilized DLL4 suppressed the expression of VEGFR2 (FIG. 7g).
Therefore, while VEGF can act upstream of DLL4/Notch pathway, DLL4/Notch
is capable of fine-tuning the response through negatively regulating
VEGFR2 expression.

[0466]Besides the increase in EC proliferation, antagonizing DLL4/Notch
caused a dramatic morphological change of EC sprouts in fibrin gel. The
multicellular lumen-like structures were mostly absent (FIG. 8a),
suggesting defective EC differentiation. In the Mab YW26.82-treated
retinas, the characteristic pattern of radially alternating arteries and
veins was severely disrupted. Anti-α smooth muscle actin (ASMA)
staining, which is associated with the retinal arteries, was completely
absent (FIG. 8c). This observation was remarkably similar to the
defective arterial development in DLL4+/- embryos. These findings, from
different angles, highlighted the essential role of DLL4/Notch in
regulating EC differentiation.

[0468]To directly address the possible role of DLL4/Notch signaling during
tumor angiogenesis, we investigated the impact of blocking DLL4 on tumor
growth in preclinical tumor models (FIGS. 9a-d). In HM7, Colo205 and
Calu6 xenograft tumor models (FIGS. 9a-c), YW26.82 treatment was
initiated after tumor establishment (≧250 mm3 in tumor size).
In all three models, a separation in growth rates between the control and
treatment groups became evident three days after dosing. The tumor volume
of the treatment group remained static over two weeks of treatment. In
addition to subcutaneous tumors, anti-DLL4 Mab also inhibited tumors
growing in mouse mammary fat pads. In the MDA-MB-435 tumor model,
treatment was started 14 days post tumor cell injection. A difference in
tumor growth curves between the control and treatment groups was evident
within six days after dosing and became increasingly significant as
treatment continued (FIG. 9d).

[0469]We also investigated the impact of blocking DLL4 and/or VEGF on
numerous tumor growth in preclinical tumor models (FIGS. 9e-f; i-p). In
MV-522 and WEHI3 xenograft tumor models, YW26.82 treatment and/or
anti-VEGF treatment was initiated after tumor establishment (250 mm3
in tumor size). In the MV-522 model, both YW26.82 and anti-VEGF treatment
inhibited tumor growth individually, but the combination of the two
treatments was most effective. In the WEHI3 model, anti-VEGF treatment
showed no effect on tumor growth whereas treatment with YW26.82 showed
significant inhibition of tumor growth. In the SK-OV-3×1, LL2, EL4,
H1299, SKMES-1, MX-1, SW620 and LS174T models, YW26.82 treatment (5
mg/kg, IP, twice weekly) and/or anti-VEGF treatment (5 mg/kg, IP, twice
weekly) was administered after establishment of the tumors. In each of
these models, YW26.82 treatment inhibited tumor growth alone.
Furthermore, in all of these models where the combination was tested,
YW26.82 exhibited enhanced efficacy in combination with anti-VEGF.

[0470]In light of the tumor growth inhibition, we used the EL4 mouse
lymphoma tumor model for vascular histology studies. We found that
anti-DLL4 Mab treatment resulted in a dramatic increase in endothelial
cell density (FIG. 9g). In contrast, anti-VEGF had a completely opposite
effect (FIG. 9g), although both treatments exhibited similar efficacy in
this model.

[0472]A major concern about global inhibition of Notch is that it may be
deleterious, given the pleiotropic roles of Notch signaling in regulating
the homeostasis of postnatal self-renewal systems. For instance, Notch
signaling is required to maintain undifferentiated crypt progenitor cells
in intestines (van Es, J. H. et al. Nature 435, 959-63 (2005); Fre, S. et
al. Nature 435, 964-8 (2005)). Indeed, γ-secretase inhibitors,
which would indiscriminately block all Notch activities, cause unwanted
side effects in rodents duo to a massive increase in goblet cells within
the crypt compartment (Milano, J. et al. Toxicol Sci 82, 341-58 (2004);
Wong, G. T. et al. J Biol Chem 279, 12876-82 (2004)). We examined the
small intestines of mice treated with anti-DLL4 Mab by
immunohistochemistry analyses. In contrast to DBZ treatment, no
differences in epithelial crypt cell differentiation or proliferation
profiles were identified between anti-DLL4 Mab and control groups after
six weeks of treatment (FIG. 10). Furthermore, anti-DLL4 Mab did not
alter the expression of the Notch target gene HES-1 in the rapidly
dividing transit amplifying (TA) population (FIG. 10). These results
support the notion that DLL4/Notch signaling is largely restricted to the
vascular system.

Example 14

Treatment with Anti-DLL4 Antibody Does Not Impact Adult Retinal
Vasculature

[0473]While blocking of DLL4 had a profound impact on the retinal vascular
development in neonatal mouse, administration of anti-DLL4 antibody has
no visible impact on adult retinal vasculature (FIG. 8d). Thus,
DLL4/Notch signaling is critical during active angiogenesis, but plays a
less important role in normal vessel maintenance. In agreement with this
notion, during the course of anti-DLL4 Mab treatment, no apparent weight
loss or animal death was observed in tumor-bearing mice when dosed at 10
mg/kg twice per week for up to 8 weeks. In tumor models, anti-DLL4 Mab
and anti-VEGF exhibit opposite effects on tumor vasculature, suggesting
non-overlapping mechanisms of action.

[0474]Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, the descriptions and examples should not be construed as
limiting the scope of the invention.